CN104114696B

CN104114696B - Processes using VLPs with capsids resistant to hydrolases

Info

Publication number: CN104114696B
Application number: CN201280062447.0A
Authority: CN
Inventors: 胡安·彼得罗·亨伯图·阿汉塞特; 胡安·P·阿汉塞特; 金伯利·德莱尼; 凯思琳·B·霍尔; 尼纳·萨默斯
Original assignee: Apse Inc
Current assignee: Ribonucleic Acid Renaissance Co ltd; Apse Inc
Priority date: 2011-12-21
Filing date: 2012-12-21
Publication date: 2017-05-03
Anticipated expiration: 2032-12-21
Also published as: AU2017261590A1; CA2860310A1; US20140302593A1; US9181531B2; US20130167267A1; CN107164376A; EP2798059A2; AR089440A1; EP4019636A1; WO2013096866A3; CN107164376B; HK1203082A1; MX2014007516A; JP2018038437A; WO2013096866A2; JP6332879B2; BR112014012162A2; IL232662B; BR112014012162B1; EP2798059A4

Abstract

Novel processes and compositions are described which use viral capsid proteins resistant to hydrolases to prepare virus-like particles to enclose and subsequently isolate and purify target cargo molecules of interest including nucleic acids such as siRNA's and shRNA's, and small peptides.

Description

Using the method for the VLP with the capsid to hydrolytic enzyme resistant

With cross reference to related applications

U.S. Provisional Application No. 61/578,706 that patent application claims were submitted on December 21st, 2011, in 2012 U.S. Provisional Application No. 61/607,900 and the U.S. Provisional Application No. 61/ submitted on June 19th, 2012 that March 7 submitted to 661,688 priority, here is incorporated to the entire disclosure of the U.S. Provisional Application by reference.

Sequence table is incorporated to

The complete content of the sequence table of the paper copy and computer-reader form on floppy disk of " sequence table " is quoting Mode be expressly incorporated herein, the floppy disk contains the file for being named as 450061_SequenceListing_ST25.txt, the text Part size is 77 kilobytes and creates on December 20th, 2012.

Technical field

The present invention relates to virus-like particle, and viral capsid is more particularly to used as method and the combination of nano container Thing, the nano container are used to produce, separate and purification of heterologous nucleic acid and protein.

Background technology

Virus-like particle (VLP) is the expression by some virus structural protein matter and part is derived from the granule of virus, institute Virus structural protein quality structure is stated into peploses and/or capsid, but VLP does not contain viral genome and is noninfective.VLP is For example originating from hepatitis B viruss and some other viruses, and have been used for studying in Virus assemble and vaccine development.

Viral capsid is made up of at least one protein, and several copies of the protein are assembled to form capsid.At some In virus, viral capsid is covered by peploses.Cell membrane of such peploses by viral glycoprotein and infected host Part constitutes, and makes viral capsid and otherwise interact therewith macromole shielding.Capsid is generally said to be makes nucleic acid shell Body, the nucleic acid coding viral genome also encode lasting required protein of the virus in natural surroundingses sometimes.In order to The viral genome of virus enters new host, and capsid must be disassembled.Such dismounting generally is being used to degrade its own by host And occur under conditions of Exterior component, and most commonly it is related to proteolysiss.Virus utilizes normal host process such as albumen Enzymolysis degraded is discharged with activating the key component in its cycle, i.e. capsid dismounting and genome.

Therefore not surprisingly, document had not previously described the capsid of the hydrolytic enzyme resistant to acting on peptide bond.Its Be a part compared with larger protein very limited number some specific peptide sequences known to be anti-slightly to some protease Property, but most of peptide sequence is not then.The virus of opposing proteolysiss is reported, but these have been peplos disease Poison, wherein capsid are shielded by peploses.In this viroid, capsid is not contacted with the protease, i.e., they are exempted from by shielding By the albumen enzyme effect.Therefore, viral capsid proteolysiss stability in such cases effect (if there is Words) it is unknown.

In the extensive manufacture of recombinant molecule such as protein, ultrafiltration is used for removing less than target generally in purification step The molecule of protein, so as to cause which to separate.Purification process also generally relates to precipitation, solvent extraction and crystallization technique.These points It is inherently simple and inexpensive from technology, because being contrasted with chromatography, they are not based on surface interaction, and It is to be interacted based on body.However, these technologies are usually limited to the application to single system, and need to specify for every Plant the different condition group of protein and expression system.Every kind of target recombinant protein is presented unique binding interactions in addition Group, so that its separation process is unique and complicated.Using these simple separation processes for recombinant protein point It is therefore very low from efficiency.

Nucleic acid includes that siRNA and miRNA major parts are manufactured using chemical synthesis process.Due to required a large amount of steps and Tend to the cost of the complexity and manufacture system of the reaction of technical difficulty, these methods are usually complicated and high cost. In addition, the synthetic agent being related to is expensive, and therefore large-scale production be easily detected by simply increasing batch size to obtain.

The content of the invention

In one aspect, this disclosure provides virus-like particle (VLP), which includes at least one heterologous goods of inclosure (cargo) capsid of molecule and packaging sequence.VLP can also be comprising at least one ribozyme enclosed by capsid.Heterologous cargo molecule Oligonucleotide or oligoribonucleotide can be included.VLP can include one or more ribozyme, and the flank of ribozyme can be packaging Sequence and oligoribonucleotide, to form nucleic acid construct.VLP can include multiple nucleic acid constructs.Comprising few ribonucleotide In the VLP of acid, oligoribonucleotide can be the short rna selected from siRNA, shRNA, sshRNA, lshRNA and miRNA.VLP can Comprising at least two ribozymes, wherein every kind of ribozyme selects an end for cutting short rna.VLP can also be comprising by least 1-100 The joint of individual nucleotide composition, wherein U of the joint comprising at least 40% A or at least 40%, the few ribose core of joint connection Thuja acid and packaging sequence or oligoribonucleotide and ribozyme.Ribozyme may be selected from such as hammerhead ribozyme and hepatitis D virus core Enzyme.Hammerhead ribozyme can be with the continuous nucleotide group complementary with least 6 continuous nucleotides of oligoribonucleotide Hammerhead ribozyme variant.Alternatively, ribozyme can be can cut which is connected with oligoribonucleotide prominent Modification hepatitis delta virus ribozyme, its speed for wild type hepatitis delta virus ribozyme speed at most about 50%.Such mutation Type HDV ribozyme can have be selected from SEQ ID No：The nucleotide sequence of 10-18.

Capsid comprising wild-type virus capsid or capsid protein matter, the open country may include according to the VLP of present disclosure Raw type viral capsid is resistance to the hydrolysis being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4, the capsid protein matter and wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence is with least 15%, at least 16%, at least 21%, extremely Few 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence is same One property, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.The capsid can be comprising with SEQ ID NO:The wild type enterobacteria phage MS2 capsid protein matter of 3 aminoacid sequence.

The heterologous cargo molecule of peptide or polypeptide can be included according to the VLP of present disclosure.VLP can also be different comprising being coupled The oligonucleotide joint of source goods peptide or peptide molecule and viral capsid.Oligonucleotide joint can be the widow comprising ribozyme sequence Ribonucleotide.Alternatively, heterologous cargo molecule may include the bimolecular goods comprising difunctional polynucleotide Molecule, the difunctional polynucleotide include the first fit sequence and the second fit sequence, the first fit sequence-specific With reference to the bioactive small molecule with about 1,500Da or less molecular weight, the second fit sequence is used to combine capsid Packaging sequence.VLP can also be comprising the bioactive small molecule combined with the first fit sequence.Bioactive small molecule can be included Herbicide or insecticide, the herbicide or insecticide may be selected from such as atrazine, Acetamiprid thimet (acetamipridphorate), Profenofos, isocarbophoss and omethoate (omethoateas).

In yet another aspect, this disclosure provides the nucleic acid construct comprising nucleotide sequence, the nucleotides sequence Row coding short rna, ribozyme and packaging sequence.Short rna may, for example, be siRNA or shRNA.Nucleic acid construct can also include 4- The connection nucleotide sequence of 100 nucleotide, in the nucleotide at least 40% be A or at least 40% be T, the wherein connection core The flank of nucleotide sequence is packager code sequence and short rna coded sequence.Nucleic acid construct can also include 4-100 nucleotide Connection nucleotide sequence, in the nucleotide at least 40% be A or at least 40% be U, the wherein side of the connection nucleotide sequence The wing is ribozyme and short rna coded sequence.The flank of ribozyme sequence can be short rna and packaging sequence.Present disclosure also includes Carrier comprising any such nucleic acid construct, and the host cell comprising examples of such carriers, and by examples of such carriers stable conversion Host cell.Host cell can be bacterial cell, such as but not limited to escherichia coli (Escherichia coli) cell, Plant cell, mammalian cell, insect cell, fungal cell or yeast cells.Host cell can also be stable by Second support Transfection, second nucleotide sequence of the Second support comprising encoding virus coat, the viral capsid is to by peptide bond hydrolysis enzyme The hydrolysis of the other catalysis of EC 3.4 is resistance.The virus protein of second nucleotide sequence codified such as encoding virus coat, institute State viral capsid and wild type enterobacteria phage MS2 capsid protein matter (SEQ ID NO:3) aminoacid sequence has at least 40% sequence iden, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.Core as described herein Acid con-struct also codified wild type enterobacteria phage MS2 capsid protein matter (SEQ ID NO:3).In such nucleic acid construct Ribozyme can be such as hammerhead ribozyme, with the continuous nucleotide group complementary with least 6 continuous nucleotides of short rna Hammerhead ribozyme variant, hepatitis delta virus ribozyme can cut its saltant type hepatitis D virus being connected with short rna Ribozyme, its speed for wild type hepatitis delta virus ribozyme speed at most 50%.Non-limiting but exemplary mutations type HDV core Enzyme has selected from SEQ ID No:The nucleotide sequence of 10-18.Present disclosure is also comprising inverted with containing core described herein The plant of acid con-struct or plant tissue, and the seed or offspring of such plant or plant tissue, the wherein seed or offspring are wrapped Containing nucleic acid construct.

In yet another aspect, this disclosure provides comprising following compositionss：A) each self-contained inclosure is at least one Multiple virus-like particles of the viral capsid of heterologous cargo molecule；And b) for present in compositionss per 100 grams of capsids with little In 4 grams amounts exist one or more product of cell lysis, wherein the product of cell lysis selected from protein, polypeptide, peptide and Its any combinations.In the composition, hydrolysis of the capsid for example to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.Clothing Shell can include such capsid protein matter, itself and wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence Row are with least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%th, at least 56%, at least 59% or at least 86% sequence iden, and to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 Hydrolysis is resistance.The capsid can include wild type enterobacteria phage MS2 capsid protein matter (SEQ ID NO:3).In the group In compound, heterologous cargo molecule can include the oligonucleotide which can be oligoribonucleotide.Oligoribonucleotide for example can be selected From siRNA, shRNA, sshRNA, lshRNA and miRNA.In the composition, each virus-like particle can also include at least one Ribozyme is planted, the wherein flank of the ribozyme is packaging sequence and oligoribonucleotide, to form nucleic acid construct, and each virus Sample granule can include multiple nucleic acid constructs.In the VLP of such composition, ribozyme can be such as hammerhead ribozyme, have The hammerhead ribozyme variant of the continuous nucleotide group complementary with least 6 continuous nucleotides of short rna, hepatitis D virus core Enzyme can cut its saltant type hepatitis delta virus ribozyme being connected with short rna, and its speed is wild type hepatitis D disease At most the 50% of malicious ribozyme speed.Non-limiting but exemplary mutations type HDV ribozyme has selected from SEQ ID No:The core of 10-18 Acid sequence.VLP in such composition can also include the connection nucleotide sequence of 4-100 nucleotide, in the nucleotide extremely Few 40% be A or at least 40% be T, wherein the flank of the connection nucleotide sequence is packager code sequence and short rna code sequence Row, or the connection nucleotide sequence of 4-100 nucleotide, in the nucleotide at least 40% be A or at least 40% be U, wherein The flank of the connection nucleotide sequence is ribozyme and short rna coded sequence.The flank of ribozyme sequence can be short rna and packaging sequence Row.VLP in such composition can include the heterologous cargo molecule of peptide or polypeptide.Such VLP in compositionss can also be wrapped Containing the oligonucleotide joint for being coupled heterologous cargo molecule and viral capsid.Oligonucleotide joint can be the widow comprising ribozyme sequence Ribonucleotide.In such composition, product of cell lysis can be less than 0.5 gram, the amount less than 0.2 gram or less than 0.1 gram Exist.

In yet another aspect, this disclosure provides for separating the method with purification target cargo molecule, the method Including：A () obtains the full cell lysate comprising multiple virus-like particles (VLP), each self-contained inclosure of the virus-like particle The capsid of at least one target cargo molecule, the wherein capsid are resistances to the hydrolysis being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 's；B () implements the hydrolysis using the other EC of peptide bond hydrolysis enzyme 3.4 to VLP, its time and condition be enough to cut full cell lysis Present in thing but not by capsid enclose per 100 in indivedual polypeptides at least 60, at least 70, at least 80 or at least 90, Simultaneously before such hydrolysis present in full cell lysate per at least 60 in 100 capsids, at least 70, at least 80 or at least 90 keep complete after hydrolyzing.In the method, capsid can each self-contained such viral capsid proteins matter, itself and wild type Enterobacteria phage MS2 capsid protein matter (SEQ ID NO:3) aminoacid sequence is with least 15%, at least 16%, at least 21%th, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% Sequence iden, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.The capsid can each self-contained open country Raw type enterobacteria phage MS2 capsid protein matter (SEQ ID NO:3).In the method, heterologous cargo molecule can comprising which Being the oligonucleotide or peptide or polypeptide of oligoribonucleotide.Oligoribonucleotide can be selected from siRNA, shRNA, SshRNA, lshRNA and miRNA.In the method, each virus-like particle can also include ribozyme, and the flank of the wherein ribozyme is Packaging sequence and oligoribonucleotide, to form nucleic acid construct.The method may also include purification capsid after hydrolyzing.Purification can Including at least one of liquid-liquid extraction step, crystallisation step, fractional precipitation step and ultrafiltration step.Present disclosure is also wrapped Containing the compositionss produced by this class method.

In yet another aspect, this disclosure provides after intracellular production target molecule in host cell, for protecting The method not hydrolyzed by the target molecule protected in full cell lysate, the method include：A () is selected to by the other EC of peptide bond hydrolysis enzyme The hydrolysis of 3.4 catalysis is the viral capsid of resistance；(b) with first vector and Second support stable transfection host cell, described One carrier forms the nucleotide sequence of the virus protein of viral capsid comprising coding, and the Second support includes the core comprising ribozyme Acid sequence, the flank of the ribozyme is packaging sequence and siRNA sequence；(c) make cell maintain be enough to make it is inverted thin The time of the capsid of cellular expression and assembling shell ribozyme, the flank of the ribozyme was packaging sequence and siRNA sequences with the conditions of Row.In the method, capsid can each self-contained such viral capsid proteins matter, itself and wild type enterobacteria phage MS2 clothing Glutelin matter (SEQ ID NO:3) aminoacid sequence is with least 15%, at least 16%, at least 21%, at least 40%, at least 41%th, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence iden, and it is right The hydrolysis being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.

In yet another aspect, this disclosure provides enclosing the VLP's of at least one heterologous cargo molecule for purification Method, the method include：A () obtains the cell lysate comprising multiple VLP；B () makes cell lysate that foot is contacted with protease To hydrolyze time and the condition of the product of cell lysis in addition to VLP, to form hydrolyzate；(c) separate from hydrolyzate VLP.Step (c) may include that (i) performs the first precipitation for using ammonium sulfate, be subsequently the first centrifugation, to obtain the first precipitate With the first supernatant；(ii) the second precipitation using ammonium sulfate is performed to the first supernatant, is subsequently the second centrifugation, to obtain The VLP of second precipitate, wherein second precipitate comprising at least about 70%, 80% or 90% by weight.Step (c) can be wrapped Include (i) and perform the first precipitation for using ethanol, be subsequently the first centrifugation, to obtain the first precipitate and the first supernatant；With (ii) the second precipitation using ammonium sulfate is performed to the first supernatant, is subsequently the second centrifugation, to obtain the second precipitate, wherein VLP of second precipitate comprising at least about 70%, 80% or 90% by weight.Step (c) may include to surpass hydrolyzate Centrifugation, to obtain comprising the precipitate of the VLP of at least about 70%, 80% or 90% by weight.In the method, VLP can be each Self-contained is the capsid of resistance to the hydrolysis that is catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4, and the capsid can include such capsid Protein, itself and wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence with least 15%, extremely Few 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence iden, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.VLP can Each self-contained wild type enterobacteria phage MS2 capsid protein matter (SEQ ID NO:3).In the method, step (b) can be about At least about 30 minutes are performed at 37 DEG C.Before the process may additionally include step (b), cell lysate and nuclease, amylase are made At least about 30 minutes are contacted at about 37 DEG C with least one in lipase (lypase).In the method, protease can be with The e.g. other EC 3.4 of peptide bond hydrolysis enzyme, which may be selected from such as E.C. 3.4.21.64, from streptomyces griseuses (Streptomyces Griseus protease), the protease from Bacillus licheniformis (Bacillus lichenformis), pepsin and wood Melon protease.In the method, the oligonucleoside which can be oligoribonucleotide can be included by the heterologous cargo molecule that VLP is enclosed Acid, or peptide or polypeptide.Oligoribonucleotide can be selected from siRNA, shRNA, sshRNA, lshRNA and miRNA.In the method In, VLP each can also include ribozyme as described herein, and its flank is packaging sequence and oligoribonucleotide, to form nucleic acid Construct.Oligoribonucleotide and packaging sequence can be connected by joint sequence, and the joint sequence has at least 1-100 nucleoside Acid, and comprise more than 40% A, the U more than 40% or the T more than 40%.The method passes through before may additionally include step (a) It is following to prepare cell lysate：Centrifuge cell after VLP is expressed in cell；Resuspension cell；Cell lysis and centrifuge cell splits Solution thing, to obtain supernatant, the wherein supernatant is used as the cell lysate of step (a).

In yet another aspect, this disclosure provides VLP, the VLP include at least one heterologous cargo molecule of inclosure With the capsid of packaging sequence, the wherein capsid is wild type enterobacteria phage MS2 capsid (SEQ ID NO comprising which:3) change The capsid protein matter of body.Capsid protein matter can be such capsid protein matter, and which has wild type enterobacteria phage MS2 clothing Shell (SEQ ID NO:3) aminoacid sequence, except the A residues at the 1st are lacked, and to other by peptide bond hydrolysis enzyme Outside the hydrolysis resistance of the catalysis of EC 3.4.Capsid protein matter can be such capsid protein matter, and which has wild type intestinal Bacillus phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, except the A residues at the 1st are lacked and the 2nd S residues at position are lacked, and to outside the hydrolysis resistance that is catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4.Capsid protein Matter can be such capsid protein matter, and which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid Sequence, except being lacked in the A residues at the 1st, the S residues at the 2nd are lacked and the N residues at the 3rd are lacked Lose, and to outside the hydrolysis resistance that is catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4.Capsid protein matter can be such Capsid protein matter, which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, except Y residues at 129 are lacked, and to outside the hydrolysis resistance that is catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4.Capsid Protein can be such capsid protein matter, and which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) ammonia Base acid sequence, but with single (1) aminoacid deletion in 112-117 sections, and to by the other EC of peptide bond hydrolysis enzyme The hydrolysis of 3.4 catalysis is resistance.Capsid protein matter can be such capsid protein matter, and which has wild type enterobacteria phagocytosis Body MS2 capsids (SEQ ID NO:3) aminoacid sequence, but lack with single (1) aminoacid in 112-117 sections Lose, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.Capsid protein matter can be such capsid Protein, which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but with 65-83 1-2 residue insertion in section, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.Capsid protein Matter can be such capsid protein matter, and which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid Sequence, but with the 1-2 residue insertion in 44-55 sections, and the water to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 Solution is resistance.Capsid protein matter can be such capsid protein matter, and which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but with single (1) the residue insertion in 33-43 sections, and to by peptide bond The hydrolysis of the catalysis of hydrolytic enzyme classification EC 3.4 is resistance.Capsid protein matter can be such capsid protein matter, and which has open country Raw type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but it is residual with 1-2 in 24-30 sections Base is inserted, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.Capsid protein matter can be such Capsid protein matter, which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but with Single (1) residue insertion in 10-18 sections, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance 's.Capsid can be comprising the capsid protein matter sequence monomer connected with the second capsid sequence monomer, the capsid protein matter monomer sequence Equip with the hydrolysis being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 in pairs be resistance capsid.The capsid can comprising its C-terminal by The capsid protein matter sequence monomer that 0-6 residue linkers section extends, the C-terminal of the joint section and the second capsid sequence monomer Series connection, it is all these be assembled into the hydrolysis being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 be resistance capsid.Joint section can With sequence for example-(Gly)_x, wherein x=0-6, including-Gly-；-Gly-Gly-；With-Gly-Gly-Gly-.Joint section can Being the Gly-Ser joints selected from-Gly-Gly-Ser-Gly-Gly- ,-Gly-Gly-Ser and-Gly-Ser-Gly-.The capsid Can be assembled into by peptide bond hydrolysis enzyme comprising the capsid protein matter connected with the 3rd capsid sequence monomer, the capsid protein matter The hydrolysis of the other catalysis of EC 3.4 is the capsid of resistance.The capsid can include the clothing that wherein C-terminal is extended by 0-6 residue linkers section Glutelin matter, the C-terminal of the joint section are connected with the 3rd capsid sequence monomer, all these to be assembled into by peptide bond hydrolysis The hydrolysis of the other catalysis of EC 3.4 of enzyme is the capsid of resistance.The capsid can include capsid protein matter, and the wherein capsid includes capsid Protein, wherein one or two joint sequence are-(Gly)_x, wherein x=0-6, including-Gly-；-Gly-Gly-；With-Gly- Gly-Gly-.Joint section can be selected from-Gly-Gly-Ser-Gly-Gly- ,-Gly-Gly-Ser and-Gly-Ser-Gly- Gly-Ser joints.

Such capsid protein matter be for example assembled into the hydrolysis being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 be resistance clothing Shell.For example, the capsid can be-(Gly) x- comprising wherein one or two joint sequence, the capsid protein matter of x=1, the clothing Glutelin matter be assembled into the hydrolysis being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 be resistance capsid.The capsid can be comprising wherein One or two joint sequence is-(Gly) x-, and the capsid protein matter of x=2, the capsid protein matter are assembled into by peptide bond water The hydrolysis of the other catalysis of EC 3.4 of solution enzyme is the capsid of resistance.The capsid comprising wherein one or two joint sequence can be- (Gly) the capsid protein matter of x-, x=3, the capsid protein matter are assembled into the water to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 Solution is the capsid of resistance.The capsid can include one or more coat protein sequence of its N-terminal 1-3 residue of truncate, and And the as described herein number of residues of connector area elongated segment disappearance, and which is to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 Hydrolysis is resistance.The capsid can include one or more coat protein sequence of its 1 residue of C-terminal truncate, and such as One residue of connector area elongated segment described herein, wherein the capsid is to the hydrolysis by the other EC3.4 catalysis of peptide bond hydrolysis enzyme It is resistance.The capsid can be included in the first coat protein sequence of its 1 residue of C-terminal truncate in series connection dimer, and Extend the joint section of a residue, or first and/or second housing protein sequence C-terminal wherein in series connection trimer 1 residue of truncate, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.Capsid can include such clothing Glutelin matter, which has N-terminal and a C-terminal truncate, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance 's.

Coloured picture is referred to

Application documents contain at least one photochrome.Copy disclosed in present patent application with photochrome will asked Summation pays necessary expenses and is provided by Patent Office.

Description of the drawings

Fig. 1 is optical density (OD as time go by；Solid diamond) and pH (open squares) figure, show wild type MS2 bacteriophages (ATCC numberings 15597-B1, from American Type culture center, Rockville, MD) are in its large intestine bar Breeding in bacterium host (ATCC numberings 15669).

Fig. 2 is bred and using the MS2 bacteriophages obtained after E.C. 3.4.21.64 and ultrafiltration purification in being shown in escherichia coli The gel of the SDS-PAGE analysis results of sample, show E.C. 3.4.21.64 purification obtain be purified to phage higher than 99% ( Band at 14kDa corresponds to MS2 bacteriophage coat proteins).

Fig. 3 is the gel of the SDS-PAGE analysis results of the MS2 of display portion purification, show phage degradable and The result (swimming lane 4 and 6) obtained after 1x the or 2x ultrafiltration of lysate.

Fig. 4 is the gel of the SDS-PAGE analysis results for showing the MS2 samples that purification is processed using ultrafiltration and E.C. 3.4.21.64.

Fig. 5 is the gel of the SDS-PAGE analysis results for showing MS2 samples, the MS2 samples are processed using E.C. 3.4.21.64, Precipitate in acid condition, purification is carried out using ethanol precipitation and ultrafiltration under alkalescence and acid condition.

Fig. 6 is the figure of the UV spectrum for showing MS2 samples, and the MS2 samples are processed using E.C. 3.4.21.64, in acid condition Precipitate, purification is carried out using ethanol precipitation and ultrafiltration under alkalescence and acid condition.

Fig. 7 after purification, is carried out in 1.5% agarose gel dyeed with Ethidum Eremide for the descriptions of Fig. 5 and 6 (1.2kbp is for primers F 1201_1223-R1979_ in swimming lane 1 for the chromatogram of the PCR primer for deriving from MS2 samples of chromatograph 2001,800bp for primers F 1201_1223-R1979_2001 in swimming lane 2, and 304bp is for the primer in swimming lane 3 F1401_1426-R1680_1705), show the concordance with complete MS2 bacteriophages genome.

Fig. 8 be for Fig. 5 and 6 description after purification, with control sample (open diamonds) and MS2 samples acquisition with Time past optical density (OD；Solid diamond) figure, show the sample of purification containing high infective phage of withing a hook at the end.

After Fig. 9 is shown in the MS2 capsids expression of encapsidate RNA, the gel of the SDS-PAGE analysis results of MS2 samples, The RNA codings are attached to the coat protein of 19 aggressiveness RNA hair clips of shell specificity.

Figure 10 is after purification, to carry out (in swimming lane 1 of chromatograph in 2% agarose gel dyeed with Ethidum Eremide 304bp；Leftmost swimming lane is corresponding to 1kb plus the ladder from Life Technologies), from regard to MS2 shell portions Presence or absence of the PCR primer chromatogram of the PCR inquiries of MS2 samples, the concordance with complete MS2 envelope genes is shown.

Figure 11 is to show the purification for MS2 virus-like particles (VLP), after with ethanol simple precipitation, MS2 samples The gel of SDS-PAGE analysis results.

Figure 12 is to show the purification for MS2 VLP, using E.C. 3.4.21.64 (PK) and with after ethanol simple precipitation, MS2 samples The gel of the SDS-PAGE analysis results of product.

Figure 13 is to show the purification for MS2 VLP, using composition hydrolytic enzyme (CH), with ethanol precipitation and super After filter, the gel of the SDS-PAGE analysis results of MS2 samples.

Figure 14 is to show the purification for MS2 VLP, using various hydrolytic enzyme and with after ammonium sulfate precipitation, MS2 The gel of the SDS-PAGE analysis results of sample.

Figure 15 is to show to obtain the gel of the PAGE analysis results of the RNA of the RNA of encapsidate in comfortable MS2 capsids.

Figure 16 is shown in PAGE point using the RNA products produced after hepatitis D virus (HDV) ribozyme in vitro transcription The gel of analysis result.

The PAGE of the siRNA products that Figure 17 is obtained during being shown in the in vitro transcription using long flank hammerhead ribozyme The gel of analysis result.

After Figure 18 is shown in VLP purification and RNA is separated from VLP, obtain the RNA products of the RNA of encapsidate in comfortable VLP PAGE analysis results gel.

Figure 19 is shown in VLP purification and suspension, and after being exposed to multiple protein enzyme 1 hour and incubating for 4 hours, comprising A series of gels of the SDS-PAGE analysis results of the VLP of MS2 capsids.

Figure 20 is the comparison of selected enterobacteria phage MS2 capsid protein matter.

Figure 21 is retrieved from UniProt data bases, and is used its BLAST multiple alignment with Weighting Matrices column selection, lacked What the default value of mouth point penalty etc. was compared, it is completely smooth the comparison of Viraceae (leviviridae) virus capsid protein matter sequence.

Figure 22 is 1AQ3 chain B (Leviviridae coat protein monomer) and 1QBE chain C (alloleviridae shell eggs White matter monomer) main chain superposition illustrate.

Figure 23 is 1AQ3 chain B (Leviviridae coat protein monomer) and the 1QBE chain C shown in Figure 22 The alternative view of the main chain superposition of (alloleviridae coat protein monomers) is illustrated.

Figure 24 is 1AQ3 chain B (Leviviridae coat protein monomer) and the 1QBE chain C shown in Figure 22 Another alternative view of the main chain superposition of (alloleviridae coat protein monomers) is illustrated.

Figure 25 is 1AQ3 chain B (Leviviridae coat protein monomer) and the 1QBE chain C shown in Figure 22 Another alternative view of the main chain superposition of (alloleviridae coat protein monomers) is illustrated.

Figure 26 is compared using the structure sequence of jFATCAT rigid 1AQ3,2VTU and 1QBE.

Figure 27 is retrieved from UniProt data bases, and is used its BLAST multiple alignment with Weighting Matrices column selection, lacked What the default value of mouth point penalty etc. was compared, the comparison of complete alloleviviridae virus capsid proteins matter sequence.

Figure 28 is to show to form icosahedron (isosahedral) Leviviridae and alloleviviridae capsids Other 60 s' in 180 monomers illustrates.The main chain of each monomer is represented by the colour band of different colours.Backbone hydrogen bond by Cyan line is represented.Icosahedron triad is at the center of the figure.Monomer-monomer contact is not filled with by connection flexible ring 67-81 The centre circle of the hydrogen bond outlining at tip.

Figure 29 is shown by the list contacted with icosahedral capsid (colour band is represented as brown and atroceruleous monomer backbone) 2 MS2 monomers (colour band represents color as black and nattier blue main chain) that body is surrounded illustrates. AlloleviviridaeQ β with for Leviviridae between residue 72 and 73 (red, bottom centre) two residues Disappearance.Center gap is tightly under the deletion segment (Fig. 5).Disappearance causes its slight extension.Q β disappearances at 126 (it is red, in On the left of the heart) skew from section is removed, but it is wide between neighbouring monomer lamella in the original location substantially accommodating monomer General contact.Using MS2 sequence numbers.

Figure 30 is enclosed by the monomer contacted with icosahedral capsid (colour band is represented as brown and atroceruleous monomer backbone) Around 2 MS2 monomers (colour band represents color as black and nattier blue main chain) illustrate.alloleviviridae Qβ With for Leviviridae, between residue 12 and 13, a residue at (yellow, upper left center) is inserted, from outer capsid surface Extend to the flexible ring in solvent；Connect end the chain in goods inside is extended to, between residue 53 and 54 (yellow, Lower-left center) two residues insertion；Residue insertion between residue 27 and 28 is also extending to the β in capsid goods space Fold stock (beta-strand) connector end.These insert none movement of needs in monomer folds or between neighbours.

Figure 31 is enclosed by the monomer contacted with icosahedral capsid (colour band is represented as brown and atroceruleous monomer backbone) Around 2 MS2 monomers (colour band represents color as black and nattier blue main chain) illustrate.alloleviviridae Qβ With for Leviviridae, between residue 36 and 37, a residue of (yellow, central right) is inserted.Ring is for neighbouring spiral shell The end of rotation compresses, but the residue for inserting is may extend in the central space above flexible ring immediately below.

Figure 32 is that 3 non-covalent enterobacteria phagies MS2 compressed around point of symmetry in the capsid of assembling are non-covalent Dimeric main chain colour band is illustrated, wherein all N-terminal colors are green, C-terminal color is redness.

Specific embodiment

As the chapter title used in this section and this paper entire disclosures is not intended to be restricted.

A. define

As used herein, unless the context, otherwise singulative " one ", " one kind " and " should/institute State " including plural referent.For the narration of this paper number ranges, take explicitly into account with same accuracy between the two Each insertion number.For example, it is for scope 6-9, it is considered to the number 7 and 8 in addition to 6 and 9, and for scope 6.0-7.0, bright Really consider number 6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9 and 7.0.

Unless otherwise stated, the use of "or" means "and/or".Additionally, term " including " and other forms Using not being restricted.

Unless otherwise defined herein, the scientific and technical terms being otherwise used in combination with present disclosure should be with by ability The implication that domain those of ordinary skill is generally understood that.For example, with animal described herein and cyto-anotomy, cell and tissue training Support, any nomenclature that biochemistry, molecular biology, immunology and microbiology are used in combination and technology are this area crowd institutes It is known and be usually used those.The implication and scope of term should be clear and definite；However, in the case of any potential ambiguity, Provided herein is definition have precedence over any dictionary or external definition.In addition, unless the context otherwise requires, otherwise singular references should Including plural number, and plural term should include odd number.

Be used and can be used for put into practice the chemistry of method described herein and compositionss, biochemistry, molecular biology and Broad range of routine techniquess and instrument in immunology, in the ability of those of ordinary skill in the art, and in the literature Fully describe.Such technology and instrument include that for generating technology and instrument with purification VLP the VLP is included with wild type It is or recombinant capsids are together with the VLP of one or more cargo molecule and as described herein for transformed into host organisms and expression The technology and instrument of recombinant protein and nucleic acid.See, for example, MOLECULAR CLONING, A LABORATORY MANUAL 2 editions .1989 (Sambrook et al., Cold Spring Harbor Laboratory Press)；And CURRENT (Ausubel et al. is edited PROTOCOLS IN MOLECULAR BIOLOGY, GreenePubl.Assoc., Wiley- Interscience,NY)1995.The respective disclosure of the list of references is hereby incorporated herein by.

As used herein, term " cargo molecule " refer to by capsid enclose or can by capsid enclose oligonucleotide, polypeptide Or peptide molecule.

As used herein, term " oligonucleotide " refer to by di-phosphate ester it is bonded at least two and no more than about 70 The short polymer of nucleotide, preferably more than about 55 nucleotide.Oligonucleotide can be oligodeoxynucleotide (DNA) or few core Ribotide (RNA), and it is such as, but not limited to siRNA, shRNA, sshRNA and miRNA comprising short rna molecule.

As used herein, term " peptide " refers to polymer molecule, its bottom line include by peptide it is bonded at least two Amino acid monomer, and preferably with by peptide it is bonded at least about 10, and more preferably at least about 20 amino acid monomers, and not More than about 60 amino acid monomers, preferably more than about 50 amino acid monomers.For example, the term comprising with about 10, about 20, The polymer of about 30, about 40, about 50 or about 60 amino acid residues.

As used herein, term " polypeptide " refers to including the polymer by least one bonded amino acid monomer chain of peptide Molecule, the wherein chain include at least about 70 amino acid residues, preferably at least about 80, more preferably at least about 90, and even more preferably At least about 100 amino acid residues.As used herein, the term includes protein, and which may include one or more connection Polypeptide chain, the polypeptide chain can be also combined or not combined with cofactor or other protein.Term " albumen as used herein Matter " is used interchangeably with term " polypeptide ".

As used herein, " variant " of the term for molecule is the sequence substantially class with natural or wild type molecule As sequence.For nucleotide sequence, variant includes such sequence, its can with regard to one or more sequence changes, but by In the degeneracy of genetic code, the same acid sequence of native protein is still encoded.Variant includes naturally occurring equipotential base Cause, and transformed using such as direct mutagenesises of widely-known technique in molecular biology and encode the nucleoside of native protein Acid sequence, and the sequence of polypeptide of the coding with amino acid replacement.Usually, nucleotide sequence variants of the invention with it is natural (endogenous) nucleotide sequence has at least 40%, at least 50%, at least 60%, at least 70% or at least 80% sequence iden. Present disclosure is also included has at least about 85% sequence iden, at least about 90% sequence iden, at least about 85%, 86%th, 87%, 88%, 89%, 90%91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nucleotide Sequence variants.

As what is used for giving nucleotide sequence herein, term " conservative variant " refers to the amino of coding and reference sequences The nucleotide sequence of the identical or substantially the same aminoacid sequence of acid sequence.Due to the degeneracy of genetic code, thus almost The codon codified of exceed all the time one each aminoacid, the nucleotide sequence of the closely related protein of coding can not be total to Enjoy high-caliber sequence iden.Additionally, the different biological preferred codons having for many aminoacid, and it is different biological Or or even identical biological different strains such as coli strain, can have the difference preferably passwords for same amino acid Son.Therefore, the first nucleotide sequence for encoding the polypeptide substantially the same with the second nucleotide sequence is considered as and the second nucleotide Sequence is substantially the same, even if they do not share the sequence iden of bottom line percentage ratio, or incite somebody to action that under strict conditions This hybridization.Additionally, it should be appreciated that in the case of the limited exception of the ATG of the unique codon of usually methionine, Ren Hexu Row can be modified by standard technique, to obtain functionally identical molecule, and such modification by present disclosure bag Contain.As described in hereinbelow, present disclosure especially considers the protein variants of native protein, and which has aminoacid sequence Row, the aminoacid sequence and native nucleotide sequence are with least 15%, at least 16%, at least 21%, at least 40%, at least 41%th, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence iden.

The sequence iden of aminoacid sequence or nucleotide sequence in the limited area of molecule or across full length sequence, Can be easily determined by using conventional tool known in the art and as described herein and method.For example, two aminoacid sequences or The degree of sequence identity of two nucleotide sequences is easily determined by using comparison instrument, and the comparison instrument such as NCBI is basic Local Alignment Search Tool (Basic Local Alignment Search Tool) (BLAST) (Altschul et al., 1990), which easily can derive from multiple in line source.The algorithm that optimal sequence is compared is it is well known in the art that and describing, bag Include for example in Smith and Waterman, Adv.Appl.Math.2:482(1981)；Pearson and Lipman Proc.Natl.Acad.Sci.(U.S.A.)85：In 2444 (1988).The algorithm of sequence analysis also program such as blastp, Can be readily available in blastn, blastx, tblastn and tblastx.For the purpose of present disclosure, when two nucleotide When sequence hybridizes under strict conditions each other, they also can be considered " substantially the same ".Stringent condition includes high hybridization temperature With the less salt in hybridization buffer, which is only allowed in the hybridization between highly similar nucleotide sequence.Stringent condition be sequence according to Bad property, and will be different under various circumstances, but at least about 60 DEG C of temperature is generally included, which is than to limit ion strong Degree and pH under about 10 DEG C low for the thermal melting point (Tm) of particular sequence to about 15 DEG C.Salinity is usually under pH7 about 0.02 mole.

Degree of sequence identity between two aminoacid sequences can use Karlin and Altschul (Proc.Natl.Acad.Sci.USA87:BLASTp algorithms 2264-2268,1993) are measured.Sequence iden percentage It is measured than the sequence by comparing two optimal comparisons on relatively window, wherein for the optimal comparison of two sequences, with Reference sequences (which does not include addition or lacks) compare, and the aminoacid sequence part in relatively window can be comprising addition or disappearance (i.e. breach).Percentage ratio is calculated by following：Determine the positional number that same amino acid is present in the two sequences in this place Mesh, to obtain matched position number, by matched position number divided by the total position number compared in window, and result is multiplied by 100, to obtain Percentage of sequence identity.

It would be recognized by those skilled in the art that polypeptide can be " essentially similar ", because aminoacid can be by similar amino Sour residue substitutions, and do not affect the function of mature protein.Which is that " essentially similar " peptide sequence is shared as above Sequence, in addition to not identical resi-dues can have conserved amino acid to change.Conservative amino acid replacement refers to similar The interchangeability of the residue of side chain.For example, with aliphatic lateral chain one group of aminoacid is glycine, alanine, L-Valine, bright Propylhomoserin and isoleucine；With aliphatic-hydroxy side chains one group of aminoacid is serine and threonine；With beta-branched side One group of aminoacid be agedoite and glutamine；With beta-branched side one group of aminoacid is Phenylalanine, L-Tyrosine And tryptophan；With basic side chain one group of aminoacid is lysine, arginine and histidine；With with sulfur-containing side chain one group Aminoacid is cysteine and methionine.Preferred conservative amino acid replacement group includes：Valine-lysine-isoleucine, Phenylalanine-tyrosine, Lys-Arg, alanine-valine and asparagine-glutamin.

The nucleic acid of encoded peptide, polypeptide or protein can be selected by using the screening of the derivation aminoacid sequence of given protein CDNA or genomic library are obtaining.Can using the routine operation such as the primer extension operation described in such as Sambrook et al. For detecting precursor and processing intermediate product.

The virus-like particle (VLP) being made up of the capsid for enclosing cargo molecule

Method described herein and compositionss are the results of the part of following understandings：Some viral capsids can in coming of new and Prepare in purification process and/or use, to improve the commercial operation of nucleic acid.Method described herein is using to being readily available Hydrolytic enzyme resistant recombinant viruses capsid, include protein to enclose heterologous cargo molecule such as nucleic acid, peptide or polypeptide.

Capsid can be wild type capsid or the saltant type capsid from wild type capsid, condition be when capsid with act on When at least one hydrolytic enzyme of peptide bond is contacted, capsid is shown to by the resistance for hydrolyzing enzymatic hydrolysis.It is such as interchangeable herein Use, phrase " to hydrolytic resistance " and " hydrolytic resistance " refer to any capsid, which is worked as and is present in also containing the polypeptide (polypeptide Be product of cell lysis and do not enclose in capsid) full cell lysate in when, and implement using the other EC of peptide bond hydrolysis enzyme 3.4 hydrolysis, its time and condition be enough to cut present in lysate (which is product of cell lysis and does not enclose in capsid) Per 100 in indivedual polypeptides at least 60, at least 70, at least 80 or at least 90 it is (i.e. all not enclose polypeptide at least individually 60%th, at least 70%, at least 80% or at least 90% be cut), but exist before such hydrolysis per in 100 capsids At least 60, at least 70, at least 80 or at least 90 keep complete after hydrolyzing.Hydrolysis can carry out such time period and condition, It was the cell protein before hydrolysis is carried out from the mean molecule quantity of the remaining cell protein of cell line later which be enough to make hydrolysis Mean molecule quantity less than about 2/3rds, less than about half, less than about 1/3rd, less than about a quarter or be less than About 1/5th.Method may also include remaining complete capsids after purification hydrolysis, and measure the weight of capsid and in hydrolysis With the weight of the total dried cell mass before and after purification, wherein capsid weight is divided by total dried cell mass in hydrolysis and after purification Weight be capsid weight divided by the total dried cell mass in hydrolysis and purification pre-test weight at least twice.Capsid weight It is capsid weight divided by such hydrolysis and purification pre-test divided by the weight in hydrolysis and total dried cell mass after purification The weight of total dried cell mass more than at least 10 times, preferably greater than 100 times, more preferably above 1,000 times, and most preferably more than 10,000 times.

Hydrolytic enzyme is the catalyzing hydrolysis being sorted under ID EC 3 by European commission (European Commission) The enzyme of reaction.For example, the enzyme of ester linkage hydrolyzing is catalyzed with the ID started with EC 3.1.The enzyme of catalysis hydrolysis of glycoside bond has With the ID that EC 3.2 starts.The enzyme of catalysis peptide bond hydrolysis is with the ID started with EC 3.4.Which is catalytic proteins The protease of the enzyme of hydrolysis is classified using the ID started with EC 3.4, including but not limited to E.C. 3.4.21.64 and hay bar Mycoproteinase.For example, E.C. 3.4.21.64 has ID EC 3.4.21.64.Present disclosure is (in non-limitative example comprising which In) E.C. 3.4.21.64, the protease from streptomyces griseuses, the protease from Bacillus licheniformis, pepsin and Papain The VLP of enzyme resistance, and using the method and process of such VLP.

NK of International Union of Biochemistry and Molecular Biology (Nomenclature Committee of the International Union of biochemistry and Molecular Biology) it is also recommended that by enzymatic The enzyme name of reaction and classification.Recommending completely for they can be free and widely available, and for example especially can be in http:// Online access at enzyme.expasy.org and www.chem.qmul.ac.uk/iubmb/enzyme/.IUBMB is developed for The stenography that every kind of enzyme is which site of activity for which is described.The enzyme of indistinction cutting is referred to as extensive specificity.Its He is described as Xaa at the cut mode of enzyme | Yaa, wherein | cleavage site is represented, Xaa=is { by enzyme preference in the N-terminal side of cutting Residue set, and Yaa=cutting C-terminal side on by enzyme preference residue set.Some enzymes have more than that Combination demand so that description can become more sophisticated.For the enzyme of the catalysis very reaction of specificity, for example, thrombinogen is added Enzyme of the work for active enzyme thrombin, then the activity is the basis of cutting description.In some cases, the precise activity of enzyme is probably not Clearly, and in such cases, cutting result of the report for standard testing protein such as B chains insulin.As use The substitute of the enzyme of the catalysis peptide bond hydrolysis with the ID started with EC 3.4, can use extensive enzyme-specific, and which has Xaa | Yaa is preferential, and wherein the enzyme has reported P1 bags with reference to preferential Xaa, but to reference to P1' bags Yaa without preferential, and on the contrary, Wherein the enzyme has reported P1' bags with reference to preferential Yaa, but to reference to P1 bag Xaa without preferential.

Capsid can also so be selected and/or be prepared so that they can be separated with purification process using simple separation And purification, as further detailed herein.For example, capsid may be selected or genetic modification is with than week as described herein The significantly higher hydrophobicity of substrate is enclosed, to be separated into the nonpolar and water that they are simply extracted in which immiscible so as to selectivity In phase.Alternatively, the improvement ability that capsid just can be crystallized by solution-selective is selected or genetic modification.

It is possibly realized by following using the use of simple effective purification process of capsid：Some wild type capsids are selected, Or the modification of the aminoacid sequence to the protein comprising wild type capsid so that capsid is shown to as described in herein above By the resistance of the enzymatic hydrolysis of at least one hydrolysis for acting on peptide bond.Such wild type capsid such as wild type MS2 capsids Can be used in purification process, the cheap enzyme of some of which such as E.C. 3.4.21.64 or subtilisin are used for proteolysiss.It is unrestricted Property example is enterobacteria phage MS2 (SEQ ID NO:1, complete MS2 wild type genes group；SEQ ID NO:2, MS2 wild types Coat protein, DNA sequence；With SEQ ID NO:3, MS2 wild type coat protein matter, aminoacid sequence.

(SEQ.ID NO：1) complete MS2 genomes, wild type, the coat protein represented with runic：

GGGTGGGACCCCTTTCGGGGTCCTGCTCAACTTCCTGTCGAGCTAATGCCATTTTTAATGTCTTTAGCG AGACGCTACCATGGCTATCGCTGTAGGTAGCCGGAATTCCATTCCTAGGAGGTTTGACCTGTGCGAGCTTTTAGTAC CCTTGATAGGGAGAACGAGACCTTCGTCCCCTCCGTTCGCGTTTACGCGGACGGTGAGACTGAAGATAACTCATTCT CTTTAAAATATCGTTCGAACTGGACTCCCGGTCGTTTTAACTCGACTGGGGCCAAAACGAAACAGTGGCACTACCCC TCTCCGTATTCACGGGGGGCGTTAAGTGTCACATCGATAGATCAAGGTGCCTACAAGCGAAGTGGGTCATCGTGGGG TCGCCCGTACGAGGAGAAAGCCGGTTTCGGCTTCTCCCTCGACGCACGCTCCTGCTACAGCCTCTTCCCTGTAAGCC AAAACTTGACTTACATCGAAGTGCCGCAGAACGTTGCGAACCGGGCGTCGACCGAAGTCCTGCAAAAGGTCACCCAG GGTAATTTTAACCTTGGTGTTGCTTTAGCAGAGGCCAGGTCGACAGCCTCACAACTCGCGACGCAAACCATTGCGCT CGTGAAGGCGTACACTGCCGCTCGTCGCGGTAATTGGCGCCAGGCGCTCCGCTACCTTGCCCTAAACGAAGATCGAA AGTTTCGATCAAAACACGTGGCCGGCAGGTGGTTGGAGTTGCAGTTCGGTTGGTTACCACTAATGAGTGATATCCAG GGTGCATATGAGATGCTTACGAAGGTTCACCTTCAAGAGTTTCTTCCTATGAGAGCCGTACGTCAGGTCGGTACTAA CATCAAGTTAGATGGCCGTCTGTCGTATCCAGCTGCAAACTTCCAGACAACGTGCAACATATCGCGACGTATCGTGA TATGGTTTTACATAAACGATGCACGTTTGGCATGGTTGTCGTCTCTAGGTATCTTGAACCCACTAGGTATAGTGTGG GAAAAGGTGCCTTTCTCATTCGTTGTCGACTGGCTCCTACCTGTAGGTAACATGCTCGAGGGCCTTACGGCCCCCGT GGGATGCTCCTACATGTCAGGAACAGTTACTGACGTAATAACGGGTGAGTCCATCATAAGCGTTGACGCTCCCTACG GGTGGACTGTGGAGAGACAGGGCACTGCTAAGGCCCAAATCTCAGCCATGCATCGAGGGGTACAATCCGTATGGCCA ACAACTGGCGCGTACGTAAAGTCTCCTTTCTCGATGGTCCATACCTTAGATGCGTTAGCATTAATCAGGCAACGGCT CTCTAGATAGAGCCCTCAACCGGAGTTTGAAGCATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGG AACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACTCGCGTTCAC AGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCT AAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATATGGAACTAAC CATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACC CGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAGACGCCGGCCATTCAAACATGAGGATTACCCATG TCGAAGACAACAAAGAAGTTCAACTCTTTATGTATTGATCTTCCTCGCGATCTTTCTCTCGAAATTTACCAATCAAT TGCTTCTGTCGCTACTGGAAGCGGTGATCCGCACAGTGACGACTTTACAGCAATTGCTTACTTAAGGGACGAATTGC TCACAAAGCATCCGACCTTAGGTTCTGGTAATGACGAGGCGACCCGTCGTACCTTAGCTATCGCTAAGCTACGGGAG GCGAATGGTGATCGCGGTCAGATAAATAGAGAAGGTTTCTTACATGACAAATCCTTGTCATGGGATCCGGATGTTTT ACAAACCAGCATCCGTAGCCTTATTGGCAACCTCCTCTCTGGCTACCGATCGTCGTTGTTTGGGCAATGCACGTTCT CCAACGGTGCTCCTATGGGGCACAAGTTGCAGGATGCAGCGCCTTACAAGAAGTTCGCTGAACAAGCAACCGTTACC CCCCGCGCTCTGAGAGCGGCTCTATTGGTCCGAGACCAATGTGCGCCGTGGATCAGACACGCGGTCCGCTATAACGA GTCATATGAATTTAGGCTCGTTGTAGGGAACGGAGTGTTTACAGTTCCGAAGAATAATAAAATAGATCGGGCTGCCT GTAAGGAGCCTGATATGAATATGTACCTCCAGAAAGGGGTCGGTGCTTTCATCAGACGCCGGCTCAAATCCGTTGGT ATAGACCTGAATGATCAATCGATCAACCAGCGTCTGGCTCAGCAGGGCAGCGTAGATGGTTCGCTTGCGACGATAGA CTTATCGTCTGCATCCGATTCCATCTCCGATCGCCTGGTGTGGAGTTTTCTCCCACCAGAGCTATATTCATATCTCG ATCGTATCCGCTCACACTACGGAATCGTAGATGGCGAGACGATACGATGGGAACTATTTTCCACAATGGGAAATGGG TTCACATTTGAGCTAGAGTCCATGATATTCTGGGCAATAGTCAAAGCGACCCAAATCCATTTTGGTAACGCCGGAAC CATAGGCATCTACGGGGACGATATTATATGTCCCAGTGAGATTGCACCCCGTGTGCTAGAGGCACTTGCCTACTACG GTTTTAAACCGAATCTTCGTAAAACGTTCGTGTCCGGGCTCTTTCGCGAGAGCTGCGGCGCGCACTTTTACCGTGGT GTCGATGTCAAACCGTTTTACATCAAGAAACCTGTTGACAATCTCTTCGCCCTGATGCTGATATTAAATCGGCTACG GGGTTGGGGAGTTGTCGGAGGTATGTCAGATCCACGCCTCTATAAGGTGTGGGTACGGCTCTCCTCCCAGGTGCCTT CGATGTTCTTCGGTGGGACGGACCTCGCTGCCGACTACTACGTAGTCAGCCCGCCTACGGCAGTCTCGGTATACACC AAGACTCCGTACGGGCGGCTGCTCGCGGATACCCGTACCTCGGGTTTCCGTCTTGCTCGTATCGCTCGAGAACGCAA GTTCTTCAGCGAAAAGCACGACAGTGGTCGCTACATAGCGTGGTTCCATACTGGAGGTGAAATCACCGACAGCATGA AGTCCGCCGGCGTGCGCGTTATACGCACTTCGGAGTGGCTAACGCCGGTTCCCACATTCCCTCAGGAGTGTGGGCCA GCGAGCTCTCCTCGGTAGCTGACCGAGGGACCCCCGTAAACGGGGTGGGTGTGCTCGAAAGAGCACGGGTGCGAAAG CGGTCCGGCTCCACCGAAAGGTGGGCGGGCTTCGGCCCAGGGACCTCCCCCTAAAGAGAGGACCCGGGATTCTCCCG ATTTGGTAACTAGCTGCTTGGCTAGTTACCACCCA

Although, it is surprising that lacking peplos, not modified wild type MS2 capsids are to 3.4 water of plurality of classes EC Solution enzyme is resistance, including but not limited to E.C. 3.4.21.64 and subtilisin so that can be prepared by full cell lysate and can be contained There are the highly purified capsid compositionss of cargo molecule.Correspondingly, this disclosure provides the VLP comprising viral capsid, institute State capsid of the viral capsid comprising wild type MS2 capsid proteins matter, and/or with the shared homology of wild type MS2 capsid proteins matter Protein, the viral capsid encapsidate cargo molecule.Cargo molecule may include one or more heterologous nucleic acids, peptide, polypeptide or Protein.May then use that classification EC3.4 hydrolytic enzyme, after the hydrolysis step from full cell lysate separate and purification these VLP, to produce highly purified VLP composition, such as by weight at least 60%, at least 70%, at least 80% or at least 85% VLP.Take explicitly into account with based on VLP weight at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% and The compositionss of 98% purity.

Present disclosure is included comprising following compositionss：A) multiple virus-like particles, its each self-contained wild-type virus Capsid and the heterologous cargo molecule of at least one target enclosed in wild-type virus capsid；And it is b) every for present in compositionss 100 grams of capsids, with less than 40 grams, less than 30 grams, less than 20 grams, less than 15 grams, less than 10 grams, and preferably smaller than 9,8,7,6,5, 4th, 3, more preferably less than 2 grams, and one or more product of cell lysis that 1 gram of amount is present even more preferably is less than, wherein this is thin Cellular lysate product is selected from protein, polypeptide, peptide and its any combinations.Subsequently, cargo molecule can be easily harvested from capsid.Phase Ying Di, such composition are what is be highly desirable to for all applications for wherein needing high-purity and/or high efficiency.

Hydrolytic enzyme resistant capsid can be used to enclose different types of cargo molecule as described herein, to form virus-like Grain.Cargo molecule can be but not limited to any one or more of oligonucleotide or oligoribonucleotide (DNA, RNA, LNA, PNA, siRNA, shRNA, sshRNA, lshRNA or miRNA, or any widow comprising any kind of non-naturally occurring nucleic acid Nucleotide), any peptide, polypeptide or protein.Which is that the cargo molecule of oligonucleotide or oligoribonucleotide can be enclosed in capsid, Use that is adjoint or being not accompanied by joint.Capsid is can trigger for example, with the presence of nucleotide goods such as oligoribonucleotide, By capsid protein matter self-assembly.In non-limitative example, capsid as described herein can enclose the heterologous RNA chains of target, for example The heterologous RNA chains of target containing altogether 1,800-2,248 ribonucleotide, gather including 19 from enterobacteria phage MS2 Body packaging site (packsite), such RNA chains by plasmid transcription detached with the plasmid of encoding capsid protein matter, such as by Wei, Et al. Y. (2008) J.Clin.Microbiol.46:1734-1740 descriptions.

RNA interference (RNAi) is that, by the phenomenon of short rna molecule such as siRNA molecule mediation, the short rna molecule can be used for The Selective depression of target genes of interest, and with the multiple application in biotechnology and medical science.For example, short rna molecule Can be used for the specific purpose gene in target biology, phenotype is wished to obtain.However, short rna molecule includes siRNA by being referred to as RNase all over easily degrading in enzyme.Capsid capsid protective housing RNA for example described herein does not receive enzymatic degradation.However, Capsid can enclose the RNA chains containing one or more ribozyme as described herein, and the ribozyme is Self cleavage ribozyme (cis work With), or in some cases, the key (trans-acting) in other RNA can be cut.One or more ribozyme is may include for example, One or more RNA sequence is cut with specificity, to produce particularly customized RNA molecule, such as but not limited to siRNA molecule. For example, the variant of hammerhead shape and hepatitis delta virus ribozyme is known, and can be used to cut long RNA sequence.In the disclosure Appearance describes the new VLP comprising capsid, and the capsid encapsidate is attached to one or more core of packaging sequence as above Enzyme (has the RNA sequence of strong affinity) to the inwall of capsid, and for cutting short rna from the packaging sequence for being attached to ribozyme The ribozyme of sequence.

Present disclosure therefore also comprising ribozyme separate from for encapsidate its one or more packaging sequence it is short or The new application of tiny RNA sequence (such as siRNA, shRNA, sshRNA and miRNA sequence).It should be appreciated that unless the context otherwise Illustrate, otherwise term short rna includes folder (stem ring) RNA sequence of the short single-stranded and bob with double-strand stem and single-stranded loop or hair clip. Short rna is with appointing less than 30 nucleotide, preferably more than 25 nucleotide and more preferably no more than 22 nucleotide What RNA single strand；Or with stem less than 30 nucleotide bases to, preferably more than 25 nucleotide bases to it is more excellent Hairpin RNA of the choosing less than the stem of 22 nucleotide bases pair.

Challenge in using the ribozyme to such short rna sequence is separated from packaging sequence for high activity is that ribozyme can Such rapid operation, before RNA encapsidates are realized, short rna to be discharged from packaging sequence.Further it has been found that short rna is for example The three dimensional structure of siRNA or hair clip packaging sequence may interfere with the appropriate function of ribozyme.These problems can be overcome by following：1) make With the ribozyme mutant for confirming slower activity speed, to avoid, before short rna encapsidate is realized, discharging short from packaging sequence RNA, and/or the nucleotide number of Watson-Crick pair in 2) increasing ribozyme, is formed with short rna.In addition, transacting ribozyme can The RNA percentage ratios increased as short rna encapsidate in VLP are advantageously used in, if the flank of one or more short rna sequence Be not complete ribozyme, but which be the shorter sequence of transacting ribozyme target, the shorter sequence also encapsidate to identical In VLP.

One or more short rna sequence can also encapsidate to or the viral capsid of wild type or genetic modification in, the disease Malicious capsid is modified, to insert outside peptide tag, protein or drug molecule is delivered to particular kind of cell.It is wild Raw type capsid also can be inherited modification, to insert the outside peptide sequence of the part for serving as some surface protein cell receptors, can have Sharp ground is intended to induce the short rna sequence of the RNAi in specific target cell for encapsidate.Such composition is passed than current short rna The replacement scheme manufacture simply too much, less expensive and more reliable sent.

The non-limitative example of the useful VLP that can be prepared includes the capsid for enclosing RNA chains, and the RNA chains are included：

I () at least one packaging sequence and flank are identical or not for 1-100 of single-stranded (non-hybridization) RNA sept Same siRNA, each of which single stranded RNA sept have 4-40 nucleotide (SEQ ID NO:30)；

(ii) one (1) ribozyme and single-stranded (non-hybridization) RNA sequence/siRNA, each of which single stranded RNA sequence Row are with 4-40 nucleotide (SEQ ID NO:28；HDV ribozymes)；

(iii) two (2) ribozymes/siRNA；

(iv) one (1) T7 initiation site, (1) ribozyme, (1) packaging site and one (1) turn Record termination site；Or

(v) (1) T7 initiation site, (1) packaging site and (1) translational termination site.

(vi) four (4) ribozyme/siRNA (SEQ ID NO:29).

In the VLP including one or more ribozyme, present disclosure is further contemplated and contains gained after ribozyme cuts RNA Product VLP.The T7 transcription terminators described in for example stating as follows can be used including the VLP of transcription terminator：Studier FW,Moffatt BA.(1986)J.MolBiol.May5；189(1):113-30；With R Sousa, D Patra, EM Lafer (1992)J.Mol.Biol.224:319-334.For example, following two kinds of sequences are the suitable transcription terminators of t7 rna polymerase：

CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTG(SEQ ID NO:4)

Or

TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG(SEQ ID NO:5)。

VLP can alternately enclose at least one target peptide, polypeptide or protein as described herein.When the heterologous goods of target When thing molecule is peptide, polypeptide or protein, oligonucleotide joint can be used to be coupled the heterologous cargo molecule of target and viral capsid.Its Cargo molecule for peptide, polypeptide or protein preferably uses joint in capsid intermediate package.Packaging process is by by the fit sequence of short rna The joint of row composition promotes, and the short rna is fit, and sequence is formed in coat protein and is fused to the peptide mark of target cargo molecule Connection between label.(referring to Fiedler, J. et al., RNA-Directed Packaging of Enzymes within Virus-like Particles,Angew.Chem.Int.Ed.49：9648–9651(2010)).Oligonucleotide joint can be by DNA, RNA, LNA, PNA etc. are constituted.Joint is such as 50 to 100 aggressiveness, with first end for capsid inside the shell Portion has the short sequence for being for example about 20ng of binding specificity, and in the second opposed ends for goods peptide, polypeptide or egg White matter has another sequence for being for example about 70nt of binding specificity.In addition, slow ribozyme can mix the joint being made up of RNA It is interior.For example, slow ribozyme can mix packaging sequence (being combined with coat protein) and fit (the label knot with target proteins matter Close) between.After activation, ribozyme makes coat protein separate with target proteins matter.Alternatively, such as it is described herein Capsid can be enclosed in N-terminal by peptide-labeled at least one target proteins matter, the peptide can with containing fit and capsid packaging The RNA chain Non-covalent bindings of sequence, the N-terminal label for for example such as being described by Fiedler, J. et al. (2010) and containing fit and The RNA chains of packaging sequence.

Cargo molecule can be bimolecular cargo molecule, and capsid described herein can also encapsidate bimolecular goods point Son, which may include or not include one or more ribozyme.Bimolecular cargo molecule can be included and be connected to difunctional polynucleotide It is fit.The fit sequence having using SELEX specific selections, to show that the specificity with bioactive small molecule is tied Close, the bioactive small molecule has the molecule of the preferably shorter than low-molecular-weight of 1,500Da.Difunctional polynucleotide have For the first aptamer activity with reference to low-molecular-weight biological activity cargo molecule, and for second of the packaging sequence with reference to capsid Both aptamer activities.The difunctional polynucleotide for being connected to biological activity cargo molecule are formed and subsequently may be connected to double points of goods Sub- cargo molecule.Such cargo molecule can be used for binding bioactive small molecule, and therefore make VLP be mounted with small molecule.This Therefore disclosure also includes the VLP comprising the capsid for being connected to such synthesis bimolecular cargo molecule.Can by with RNA aptamer With reference to and the example of low-molecular-weight bioactivator that is loaded in VLP include：Atrazine (herbicide), Acetamiprid thimet, Profenofos, isocarbophoss and omethoate (insecticide), such as by Sett et al. (2012) Open Journal of Applied Biosensor,1:The 9-19 page description.

The example of the low-molecular-weight bioactivator that can be loaded into by combining with RNA aptamer in VLP includes herbicide Such as 2,4-D (2,4-D dichlorphenoxyacetic acids), Mediben ((bis- chloro- 2-methoxybenzoic acid of 3,6-), N,N'-dimethyl-.gamma..gamma.'-dipyridylium (N, N'- bis- Methyl -4,4'- bipyridinium dichlorides), oryzalin (4- (dipropylamino) -3,5- dinitro benzene sulfonamide), DCMU (3- (3,4- Dichlorobenzene base) -1,1- dimethyl ureas), trefanocide (2,6- dinitro-N, N- dipropyl -4- (trifluoromethyl) benzene Amine), imazapic (- methyl -2- [4- methyl -5- oxo -4- (propyl- 2- yl) -4,5- dihydro -1H- imidazoles -2- bases] pyridine - 3- carboxylic acids), chlorine Fampridine (Aminopyralid) (4- amino -3,6- dichloropyridine -2- carboxylic acids), clopyralid (3,6- bis- Chloro-2-Pyridyle carboxylic acid), isopropyl methoxalamine (the chloro- N- of (RS) -2- (2- ethyl -6- methylphenyls)-N- (1- methoxy propyl -2- Base) acetamide), pendimethalin (the amyl- 3- bases-aniline of 3,4- dimethyl -2,6- dinitro-N-), picloram (4- amino - Tri- chloro- 2-Pyridinecarboxylic Acids of 3,5,6-), propanil (N- (3,4- Dichlorobenzene base) propionic acid amide .), Triclopyr ([(tri- chloro- 2- pyrroles of 3,5,6- Piperidinyl) epoxide] acetic acid), and atrazine (2- chloro- 4- (ethylamino) -6- (isopropylamino)-s- triazines), in particular, for example by Roberts et al. (1998) Metabolic Pathways of Agrochemicals：Part1：Herbicides and Plant Growth Regulators.Royal Society of Chemistry (Great Britain) (publication) What ISBN978-1-84755-138-2 was listed.For example, with reference to atrazine RNA aptamer by Sinha et al. (2010) Nature Chemical Biology,6:P.464-470 describe.

RNA aptamer is can be also used for reference to insecticide such as propargite (2- (4- tert-butyl benzene epoxides) cyclohexyl propyl- 2- Alkynes -1- sulfonate), chlopyrifos (O, O- diethyl O-3,5,6- trichloropyridine -2- base thiophosphates), cypermethrin, imines Sulfur phosphorus (2- dimethoxy phosphinothioyl sulphomethyls) isoindoline -1,3- diketone), Permethrin (3- phenoxy benzyls (1RS)-suitable, Trans- 3- (2,2- dichloroethylenes) -2,2- dimethyl cyclopropane carboxylic acid's esters), diazinon (O, O- diethyl O- [4- methyl -6- (propyl- 2- yl) pyrimidine -2-base] thiophosphate), parathion-methyl ((O, O- dimethyl O- (4- nitrobenzophenones) D2EHDTPA Ester), and Acetamiprid (N- [(6- chloro-3-pyridyl bases) methyl]-N'- cyano-N-methyls-ethanamidine), and antifungal such as hundred bacterium (2,4,5,6- termils), captan ((3aR, 7aS) -2- [(trichloromethyl) sulfanyl] -3a, 4,7,7a- tetra- clearly - 1,3 (2H)-diketone of hydrogen -1H- iso-indoles), Boscalid (the chloro- N- of 2- (4'- chlordiphenyl -2- bases) nicotiamide), RP-26019 (3- (3,5- Dichlorobenzene base)-N- isopropyl -2,4- dioxo alkyl imidazole -1- Methanamides), Fluoxastrobin ((2E) -2- (2- { [6- (2- cyanogen Phenoxyl) pyrimidine-4-yl] epoxide phenyl) -3- methoxy-methyl acrylates), pyraclostrobin (2- [1- (4- chlorphenyls) pyrroles Azoles -3- base epoxide methyl]-N- methoxybenzene albendazoles), cyprodinil (4- cyclopropyl -6- Methyl-N-phenyl pyrimidine -2- Amine), in particular, for example by Roberts et al. (1999) Metabolic Pathways of Agrochemicals：Part2： Insecticides and Fungicides.Royal Society of Chemistry (GreatBritain) (publication) What ISBN978-1-84755-137-5 was listed.For example, it has been described that with reference to Acetamiprid, thimet, Profenofos, isocarbophoss and Omethoate it is fit, such as by Sett et al. (2012) Open Journal of Applied Biosensor, 1:The 9-19 page example Show, the DNA aptamer built using the mode similar to RNA aptamer is built using SELEX.

These herbicides, insecticide or antifungal are bioactive small molecules, i.e., with preferably shorter than 1,500Da's The molecule of low-molecular-weight.Due to its small size, the capsid of their permeable VLP for forming present disclosure, such as by Wu et al. (2005)Delivery of antisense oligonucleotides to leukemia cells by RNA bacteriophage capsids,Nanomedicine：Nanotechnology,Biology and Medicine,1:The 67-76 page of illustration.After such VLP is formed, before purification or after, these small bioactive molecules are added in disclosure In the VLP of appearance, the VLP encapsidates are fit using SELEX designs, to combine small bioactive molecules.For example by by its In adding VLP solution, and time of 30 minutes to 10 hours is for example incubated at room temperature, complete these small bioactive molecules Addition.These small bioactive molecules by via on granule axis of symmetry hole diffusion and enter VLP, and due to its with Enclose fit combination and be retained in inside.For the suitable solvent scope that is loaded into small bioactive molecules in VLP from pole Such as water and water-ethanol admixture of property is to nonpolar such as isobutyltrimethylmethane., toluene, dichloromethane or chloroform.Using nonpolar Solvent is used for VLP and dissolves for example such as by Johnson et al. (2006), Solubilization and stabilization of bacteriophage MS2 in organic solvents,Biotechnology and bioengineering, 2007.97(2)：The 224-34 page description complete, which is by means of surfactant such as dioctylis sulfosuccinas natricuses.Non-polar solven is used to fill The purposes for carrying small bioactive molecules is preferably as its dissolubility in polar solvent is in most of the cases very weak.

The VLP of both encapsidate siRNA and small bioactive molecules is realized between two kinds of bioactive ingredients wherein It is preferred in the application of cooperative effect, for example targeted plants, insecticide or funguses are resistances to small bioactive molecules wherein In the case of.In such cases, siRNA is designed as targeting and gives small bioactive molecules resistance to plant, insecticide or funguses Biological approach, such as by Sammons et al., Polynucleotide molecules for gene regulation in What plants, US2011/0296556 were illustrated.

Alternatively, difunctional polynucleotide codified at least one siRNA, shRNA, sshRNA, LshRNA or miRNA, and cargo molecule can be little (low-molecular-weight) protein or peptide.Correspondingly, bimolecular cargo molecule Can be with can be with reference to low molecule biological activity protein or peptide.Such bimolecular cargo molecule can include biological activity protein or Peptide, which is coupled to the polynucleotide of the siRNA or shRNA or sshRNA or lshRNA that encode at least one miRNA, and has For binding bioactive protein or the first aptamer activity of peptide cargo molecule, and for of the packaging sequence with reference to capsid Two aptamer activities.Polynucleotide are connected to protein or peptide cargo molecule, and can be connected to the packaging sequence of capsid.

Difunctional polynucleotide as above can optionally include one or more ribozyme sequence.Including bimolecular goods point Attached bag includes the VLP of difunctional polynucleotide as above can optionally include one or more ribozyme.In at least one ribozyme With bimolecular cargo molecule react with by cargo molecule cut into composition component portion include it is fit after, present disclosure is also wrapped Include the VLP of the product comprising capsid and bimolecular cargo molecule.

VLP can be assembled by any one or more of methods availalbe as described herein, and methods described is produced with assembling , the VLP of the capsid of hydrolytic enzyme resistant, described one or more cargo molecule of capsid encapsidate and optional any joint, bag Dress sequence, one or more ribozyme or label.For example, capsid and cargo molecule can in any expression system coexpression.Pass through Transfect with one or more expression vector via can be used to for examples of such carriers introducing any operation in specific cells, and stably Transfectional cell encodes one or more capsid protein matter, one kind or many to obtain the cell of one or more recombination sequence of expression The recombinant DNA for planting cargo molecule and optional any joint, packaging sequence, one or more ribozyme or label can be easily introduced into In host cell, for example bacterial cell, plant cell, yeast cells, fungal cell and zooblast (include that insecticide and suckling are dynamic Thing).

Host cell preferably has eukaryotic origin, such as plant, mammal, insecticide, yeast or fungal source, but can also make Use non-eukaryotic host cell.Suitable expression system is including but not limited to the recombinant bacteria of the coded sequence containing VLP elements The microorganism such as antibacterial (such as escherichia coli) of phage DNA, plasmid DNA or cosmid DNA expression vectors conversion.Unrestricted In property example, for the VLP using MS2 capsid protein matter, the expression in escherichia coli is suitable expression system.

Present disclosure takes explicitly into account such plant cell, and which is converted using nucleic acid construct as described herein, And express capsid coat protein, cargo molecule and optional any joint, packaging sequence, one or more ribozyme or mark Sign.The method for including plant cell and prepare transgenosis cell for transformed cells is well-known in the art.Carrier, matter Grain, cosmid, YAC (yeast artificial chromosome) and DNA section can be used for transformed cells, and as generally accepted will be including startup Son, enhancer and/or polylinker.The concrete transgenic cell for considering includes transgenic plant cells, including but not limited to derives from The cell of corn and soybean, Semen Tritici aestivi, vegetable, corn, beans, fruit tree etc., or would benefit from what VLP as described herein was introduced Any plant.It is also contemplated that the plant of transformed cells as described herein, plant tissue are derived from, and the plant or plant tissue Seed or offspring.

The expression of the VLP of assembling for example can be obtained by building at least one expression vector, and the expression vector includes The sequence of all elements of coding VLP.Sometimes use two kinds of carriers, including one or more cargo molecule of coding and optional appoint The first of the sequence of what joint, packaging sequence, one or more ribozyme or label；With the sequence including encoding capsid protein matter Second support.For generating in the illustrative methods that exemplary VLP includes siRNA, two kinds of carriers can be in host cell Coexpression, for generating VLP, as being described in further detail in example.Method and kit for for building such expression vector is this Field is it is well known that the expression vector contains coded sequence and transcription and translation control sequence.Once build, Yi Zhonghuo Variety carrier is just also using technique transfers well-known in the art to host cell, and cell subsequently maintains condition of culture Under be enough to make VLP expression and assemble the time for occurring, this uses routine techniquess.Present disclosure therefore comprising containing it is any this The host cell of class carrier, and the cell for having been converted by examples of such carriers, and the cell containing VLP.

When VLP in host cell is expressed and assembled, they can use appointing for viral purification known in the art Where method is separated and purification.For example, cell can be cracked using regular growth cracking technique and reagent, and to cell Lysate implements the hydrolysis using the other EC of at least one peptide bond hydrolysis enzyme 3.4, and the hydrolytic enzyme is such as, but not limited to protease K or subtilisin.The complete capsids being retained in cell lysate after hydrolyzing can use common protein to separate skill Art is taken out and purification.

Capsid, VLP or protein purification may also include method commonly known in the art.For example, capsid expression and After cell lysis, one or more isolated or purified steps can be implemented to the lysate of gained.Such step may include such as enzyme Promote steatolysis, DNA hydrolysis and proteolysiss step.Proteolysiss step can such as mixing using endo protease and exoproteinase Carry out with thing.For example, after cell lysis and the hydrolysis of most cells component dismounting, for example, by being extracted into suitable non-pole Property with the immiscible solvent of water in, or by being crystallized by suitable solvent, such capsid can be with surrounding substrate point with its cargo molecule From.For example, the pollutant from capsid that hydrolysis and/or proteolysiss step will be contained in cracking in substrate change into little water Soluble molecule.Hydrophobicity capsid can be subsequently extracted in double (trifluoromethyl) benzene of organic faciess such as 1,3-.Capsid, VLP or albumen The purification of matter may include for example, at least one liquid-liquid extraction step, at least one fractional precipitation step, at least one ultrafiltration step Or at least one crystallisation step.Liquid-liquid extraction may include to use for example immiscible non-aqueous non-polar solven, such as but not limited to Benzene, toluene, hexane, heptane, octane, chloroform, dichloromethane or carbon tetrachloride.Purification may include at least one crystallisation step.One Individual or multiple hydrolysing steps and the especially use of one or more proteolysiss steps, eliminate with currently used for cargo molecule Some problems that separation method is observed, which mainly originates from a large amount of and different degrees of binding interactions, the combination phase Occur between the component of the cell culture that interaction is produced wherein in cargo molecule and from them.Capsid described herein this Sample resists hydrolysing step so that the substrate for producing after hydrolyzing includes that any cargo molecule of security partition is complete with surrounding substrate Capsid, thus interrupts the troublesome binding interactions of the current purification process of interference.

After purification, capsid can be opened, to obtain cargo molecule, its can be protein as described herein or polypeptide, Peptide or nucleic acid molecules.Capsid can be opened using any one of several possible operations known in the art, including for example super Cross 50 DEG C of heated in water solution；Multigelation；Incubate together with denaturant such as Methanamide；With one or more protease one Rise and incubate；Or the combination of any one in these operations.

To hydrolytic enzyme resistant and can be used for according to present disclosure VLP and method in capsid protein matter can also be wild The variant of raw type MS2 capsid protein matter, or it is derived from wild type MS2 capsid protein matter.Capsid protein matter can comprising for example relative to At least one radical amino acid replacement of wild type MS2 capsid amino acid sequences, disappearance are inserted.Such capsid protein matter can be with It is naturally occurring variant, or can be obtained by using routine techniquess genetic modification MS2 capsid proteins matter, condition is variant Or modified capsid protein matter forms nonencapsulated capsid, which is as described herein to being urged by the other EC3.4 of peptide bond hydrolysis enzyme The hydrolysis of change is resistance.

By being made in aminoacid sequence according to the routine in physical chemistry and biochemistry and well-known principle Selected modification, can transform the MS2 capsid protein matter of the genetic modification that can be assembled into capsid as described herein to hydrolytic resistance, To produce the protein such as the holding herein and described in example hereinbelow to hydrolytic resistance.

The shape or overall folded of such as functional protein is determined by the aminoacid sequence of protein, and folds restriction egg The function of white matter, this is general knowledge.Overall folded is made up of one or more folded domains.When depositing more than a folded domain When being in overall folded, domain is typically loosely or tightly combined together along domain interfaces.Domain is folded and can be divided Folding core of the solution into close packing, well-defined Secondary structural elements, the Secondary structural elements are mainly responsible for structure The shape in domain and it is general by corner and ring group into the conformation for being more easy to mobile outer layer, the corner and ring by with fold core Interaction and include that the interaction of solvent and other protein affects with neighbouring domain and other molecules.Protein The extensive open regional data base for folding, the Protein Structure Classification (Structural of the protein structure of the solution in public domain Classification of Proteins) (SCOP) data base (Alexey G Murzin, Curr Opin Struct Biol (1996) 6,386-394) http is maintained online:At //scop.berkeley.edu, and as new solution structure is entered Public domain (Protein Data Bank (Protein Data Bank) (F.C.Bernstein, T.F.Koetzle, G.J.Williams,E.E.MeyerJr.,M.D.Brice,J.R.Rodgers,O.Kennard,T.Shimanouchi, M.Tasumi,"The Protein Data Bank：A Computer-based Archival File For Macromolecular Structures,"J.of.Mol.Biol.,112(1977)：535),http://www.rcsb.org) Data base and periodically extend.Remote in evolution family member still with same shape and closely similar function generally protects Stay at topology and/or position functionally of equal value as little as 30% identical residue.In some families, the sequence of remote member Row are with for each other unchanged as little as 20% its residue, such as Leviviridae and alloleviviridae capsids Protein.Additionally, the folding of protein and function are significantly to being tolerance via orientation or random mutation change, even core Heart residue (PeterO.S.Christopher Bauer,Sarah Braford-Goldberg,Kris Sterbenz,Joseph O.Polazzi,Maire H.Caparon,Barbara K.Klein,AlanM.Easton,Kumnan Paik, Jon A.Klover, BarrettR.Thiele and John P.McKearn (1995) J Biol Chem 270,23754- 23760；Yiqing Feng, Barbara K.Klein and Charles A.Mc Wherter (1996), J Mol Biol 259, 524-541；Dale Rennell, Suzanne E.Bouvier, Larry W.Hardy and Anthony R.Poteetel (1991) J Mol Biol222,67-87), insertion/deletion (Yiqing Feng, the Barbara of one or more residues K.Klein and Charles A.Mc Wherter (1996), J Mol Biol 259,524-541), the arrangement (Multi- of sequence functional chimeric hematopoietic fusion proteins between sequence rearranged C-mpl receptor agonists and other hematopoietic factors, US6066318), it is last via N or C Series connection (copy or other peptides or protein with its own) (the Multi-functional chimeric at end or both hematopoietic fusion proteins between sequence rearranged g-csf receptor agonists and other hematopoietic factors,US20040171115；Plevka,P.,Tars,K., Liljas,L.(2008)ProteinSci.17：173) or covalent modification, for example glycosylation, Pegylation, SUMOization or The addition of peptidyl or non-peptidyl linker affinity tag, as long as being unnecessary to maintaining the residue for folding and/or function is crucial.

According to present disclosure and as method and process any one used in VLP therefore include comprising capsid protein The VLP of matter, the capsid protein matter and wild type enterobacteria phage MS2 capsid protein matter (SEQ ID NO:3) aminoacid Sequence has at least 15%, 16%, 21%, 40%, 41%, 52%, 53%, 56%, 59% or at least 86% sequence iden, And the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.Such VLP includes for example including capsid protein matter VLP, the capsid protein matter and SEQ ID NO as above:3) with least 52% sequence iden.It is also included to be VLP comprising capsid protein matter, the capsid protein matter and SEQ ID NO:3 have at least 53% sequence iden, and which can base It is obtained as above in sheet, butNoIgnore FR capsid sequences, represent and wild type enterobacteria phage MS2 capsid protein matter (SEQ ID NO:3) 53% sequence iden.Also included is the VLP comprising capsid protein matter, the capsid protein matter and SEQ ID NO:3 have at least 56% sequence iden, this be considered as when Structural Identification be 1AQ3 (van den Worm, S.H., Stonehouse,N.J.,Valegard,K.,Murray,J.B.,Walton,C.,Fridborg,K.,Stockley,P.G., Liljas,L.(1998)Nucleic Acids Res.26：1345-1351)、1GAV(Tars,K.,Bundule,M., Fridborg,K.,Liljas,L.(1997)J.Mol.Biol.271：759-773)、1FRS(Liljas,L.,Fridborg, K.,Valegard,K.,Bundule,M.,Pumpens,P.(1994)J.Mol.Biol.244：279-290) and 2VTU (Plevka,P.,Tars,K.,Liljas,L.(2008)ProteinSci.17：1731) (above-described Protein Data Bank Identifier) when, when all three sequence considers together, only 56% sequence location is with the identical sequence for overlapping with regard to main chain Row and topology position of equal value.Also included is the VLP comprising capsid protein matter, the capsid protein matter and SEQ ID NO: 3 have at least 59% sequence iden, and this is considered as the MS2 viral capsids compared with the sequence of GA viral capsid proteins matter The sequence of protein is 59%.Also included is the VLP comprising capsid protein matter, the capsid protein matter and SEQ ID NO:3 With at least 86% sequence iden, this is considered as the MS2 viral capsid proteins matter compared with the sequence of FR capsid protein matter Sequence be 86%.Therefore VLP comprising capsid protein matter is included according to the VLP of present disclosure, based on effective structure anchor Definite proportion pair, the capsid protein matter and wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence has The sequence iden of at least 15%, 16% or 21%, and the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.

Therefore VLP can include any one in MS2 capsid proteins qualitative change body as described herein.With capsid described herein The capsid protein matter of the consistent genetic modification of protein for example can be produced by following：Build at least one capsid protein of coding At least one DNA plasmid of matter, relative to the aminoacid sequence of wild type MS2 capsid protein matter, the capsid protein matter has At least one amino acid replacement, disappearance are inserted, and are prepared multiple copies of every kind of plasmid, are used plasmid-transformed cells system；By cell Maintain be enough to the cell for making conversion expression and the time of the capsid of assembling shell nucleic acid and under the conditions of；Cell lysis are forming Cell lysate；Cell lysate is implemented using at least one peptide bond hydrolysis enzyme, the hydrolysis of classification EC 3.4；And take out in water The complete capsids being retained in after solution in cell lysate, to obtain hydrolytic enzyme at least one relative to wild-type capsid protein confrontation Capsid with increased resistance.Gained complete capsids after purification, every kind of clothing can be determined according to methods known in the art The aminoacid sequence of glutelin matter.

Special capsid described herein can be used in research and development and industrial manufacturing facility, to provide obtaining for improvement Rate, because there is the purification process used under two kinds of backgrounds identical substrate to constitute.Mainly depend on such same composition In the same cell system used in research and development and manufacture method.However, making in research and development and in the fabrication stage After proteolysiss step, due to using very different of the different cell lines in substrate composition to reduce.The feature is allowed Different cell lines used in two stages, with MIN manufacture yield punishment.

Example

Including following non-limiting examples illustrating many aspects of present disclosure.Those skilled in the art should manage Solution：Technology disclosed in following examples is represented by it is found by the applicant that the technology for well working in the practice of the invention, and And therefore can be considered the optimal way constituted for its practice.However, according to present disclosure, those skilled in the art should manage Solution can many modifications may be made in the instantiation, while same or similar result is still obtained, without departing from the model of the present invention Enclose.Therefore, example is only exemplary, and should not be construed in any way as limiting the present invention.Allowing and describing this Bright required degree, all lists of references of reference are hereby incorporated herein by.

Example A：The breeding of MS2 bacteriophages

MS2 bacteriophages (ATCC numberings 15597-B1, from American Type culture center, Rockville, MD) And its escherichia coli host (ATCC numberings 15669) derives from ATCC, and using by Strauss and Sinsheimer (1963) J.Mol.Biol7:43-54J.Mol.Biol7:The operation of 43-54 descriptions is bred.As a result mark and draw in FIG.Reacting Optical density (OD) and pH under 600nm are tracked in journey.ODi is represented tightly with the postvaccinal OD of host.Infection is complete at 2.3 hours Into.Ln (OD/ODi) marks and draws (solid diamond) in left axle, and pH is marked and drawed (open squares) in right axle.The experiment with Terminate within 5.3 hours after host's inoculation.The lysate of acquisition is centrifuged with 2,000g, and by 0.2 μm of membrane filtration, to eliminate residue Antibacterial and bacterial debris.

Example B：Using the MS2 bacteriophage purification of E.C. 3.4.21.64 and ultrafiltration

The purification of MS2 bacteriophages is carried out as follows.Sample is obtained in purge process, and SDS is run to sample PAGE is analyzed.The result of acquisition is shown in Fig. 2.

The 8mL lysates (Fig. 2, the sample in swimming lane 1) obtained at the end of example A are by 300kDa films (Vivaspin2, from Sartorius Stedim, Bohemia, NY) is filtered, and filtrate is by 100kDa membrane filtrations, by which Obtain 1mL retentates (Fig. 2, the sample in swimming lane 2).The retentate is divided into into two equal portions.To in a moiety (control), plus Enter the 206 μ L20mM CaCl with pH=7.5₂Aqueous solution.To in second moiety (protease), add with the dissolving of pH=7.5 In 206 μ L20mMCaCl₂0.15mg E.C. 3.4.21.64s (Sigma Aldrich, St.Louis, MO) in aqueous solution.Two Guan Jun exist Incubate at 37 DEG C, and after 1h, they are placed in ice-water bath.Subsequently obtain sample and analyze：In Fig. 2, swimming lane 3 Control sample, and the protease sample in Fig. 2, swimming lane 5.Subsequently use deionization (DI) water by every kind of product dilution to 2mL, And pass through 100kDa membrane filtrations.Every kind of retentate (150 μ L) is diluted to into 2mL with DI water, and again by same film mistake Filter.Dilution and ultrafiltration are repeated once to every kind of product.Subsequently obtain the sample of every kind of retentate and analyze：In Fig. 2, swimming lane 4 In control sample, and the protease sample in Fig. 2, swimming lane 6.Band at 14kDa corresponds to MS2 bacteriophages Coat protein.Band at 30kDa corresponds to E.C. 3.4.21.64.Product from control experiment obtains highly impure phagocytosis Body.Product from protease experiment is obtained containing the product with the phage higher than 99% purity.

Example C：The degraded of MS2 bacteriophages

The process of MS2 bacteriophages is carried out as follows.Sample is obtained in processing procedure, and SDS is run to sample PAGE is analyzed.The result of acquisition is shown in Fig. 3.Such as by Strauss ＆ Sinsheimer (1963) J.Mol.Biol 7:43- 54 description, by using ammonium sulfate precipitation and using Arcton 11 (freon-11) extraction, partial purification is in example A At the end of the 4mL lysates that obtain.Obtain the aqueous sample after being extracted with freon-11 and analyze (Fig. 3, in swimming lane 1 Sample).To in partially purified phage solution (130 μ L), 370 μ L20mM CaCl are added₂Aqueous solution.Mixture is made 37 Incubate at DEG C, and after 1h, be placed in ice-water bath.Subsequently obtain sample and analyze：Sample in Fig. 3, swimming lane 2 Product.Product dilution will be incubated to 2mL with deionization (DI) water, and pass through 100kDa membrane filtrations.With DI water by retentate (150 μ L 2mL is diluted to), and again by identical membrane filtration.The dilution and ultrafiltration of retentate is repeated once.Subsequently obtain and ooze remaining The sample of thing is simultaneously analyzed：Sample in Fig. 3, swimming lane 3.Only it was observed that in the weak band being less than at 10kDa, indicating phage It is degradable.

Example D：Using the MS2 bacteriophage purification of ultrafiltration.

The purification of MS2 bacteriophages is carried out as follows.Sample is obtained in purge process, and SDS is run to sample PAGE is analyzed.The result of acquisition is shown in Fig. 3.Such as by Strauss ＆ Sinsheimer (1963) J.Mol.Biol7:43- 54 description, by using ammonium sulfate precipitation and using Arcton 11 (freon-11) extraction, partial purification is in example A At the end of the 4mL lysates that obtain.Aqueous solution containing partially purified phage is diluted to 2mL by deionized water, is passed through 300kDa membrane filtrations, and filtrate obtains 150 μ L retentates by which by 100kDa membrane filtrations.Deionization (DI) water is used subsequently Retentate is diluted to into 2mL, and passes through identical 100kDa membrane filtrations.The dilution and ultrafiltration of retentate (150 μ L) repeats one It is secondary.Obtain retentate sample and analyze：Fig. 3, the sample in swimming lane 4.By 370 μ L20mM CaCl₂Aqueous solution adds retentate In (130 μ L).Mixture is incubated at 37 DEG C, and after 1h, be placed in ice-water bath.Sample is obtained subsequently simultaneously Analysis：Sample in Fig. 3, swimming lane 5.Subsequently use deionization (DI) water by product dilution to 2mL, and pass through 100kDa film mistakes Filter.Retentate (150 μ L) is diluted to into 2mL with DI water, and again by identical membrane filtration.The dilution and ultrafiltration of retentate is again It is repeated once.Subsequently obtain the sample of retentate and analyze：Sample in Fig. 3, swimming lane 6.By with less than 100kDa molecules It is clear that the protein of amount permeates the MS2 coat proteins of the 14kDa that its film retains, and indicates complete MS2 capsids Exist.The product of acquisition contains with the phage higher than 99% purity.

Example E：Using the MS2 bacteriophage purification of E.C. 3.4.21.64 and ultrafiltration

The purification of MS2 bacteriophages is carried out as follows.Sample is obtained in purge process, and SDS is run to sample PAGE is analyzed.The result of acquisition is shown in Fig. 4.

Such as by Strauss ＆ Sinsheimer (1963) J.Mol.Biol7:43-54 descriptions, by using ammonium sulfate Precipitation and using Arcton 11 (freon-11) extraction, the 4mL lysates that partial purification is obtained at the end of example A. Aqueous solution containing partially purified phage is diluted to 2mL by deionized water, by 100kDa membrane filtrations, by its acquisition 150 μ L retentates.Retentate is diluted to into 2mL with deionization (DI) water subsequently, and passes through identical 100kDa membrane filtrations.Ooze remaining The dilution and ultrafiltration of thing (150 μ L) is repeated once.Obtain retentate sample and analyze：Fig. 4, the sample in swimming lane 1.Will dissolving In 370 μ L20mMCaCl₂During 0.15mg E.C. 3.4.21.64s in aqueous solution add retentate (130 μ L).Make mixture temperature at 37 DEG C Educate, and after 1h, be placed in ice-water bath.Subsequently obtain sample and analyze：Sample in Fig. 4, swimming lane 2.Subsequently With deionization (DI) water by product dilution to 2mL, and pass through 100kDa membrane filtrations.Retentate (150 μ L) is diluted with DI water To 2mL, and again by identical membrane filtration.The dilution and ultrafiltration of retentate is repeated once.The sample of retentate is obtained subsequently Product are simultaneously analyzed：Sample in Fig. 4, swimming lane 3.The product of acquisition contains with the phage higher than 99% purity.

Example F：Using E.C. 3.4.21.64, precipitate in acid condition, ethanol precipitation is used under alkalescence and acid condition and is surpassed The MS2 bacteriophage purification of filter

The purification of MS2 bacteriophages is carried out as follows.Sample is obtained in purge process, and SDS is run to sample PAGE is analyzed.The result of acquisition is shown in Fig. 5.Such as by Strauss ＆ Sinsheimer (1963) J.Mol.Biol7:43- 54 description, by using ammonium sulfate precipitation and using Arcton 11 (freon-11) extraction, partial purification is in example A At the end of the 50mL lysates that obtain.Obtain the aqueous sample after being extracted with freon-11 and analyze (Fig. 5, in swimming lane 1 Sample).To in partially purified phage solution (1.2mL), addition is dissolved in 1.24mL20mMCaCl₂In aqueous solution 0.9mg E.C. 3.4.21.64s.Mixture is incubated at 37 DEG C, and after 1h, add 60 μ L0.2M phenylmethylsulfonyl fluorides (PMSF) Ethanol solution, with inactivated proteases K.Subsequently mixture is placed in ice-water bath.Obtain sample and analyze：In Fig. 5, swimming lane 2 In sample.With vigorous agitation, 0.68mL0.1% phosphate aqueous solutions are slowly added in ice water bath, so that the pH of liquid reaches To 4.Liquid is kept for 30 minutes at 0 DEG C, and be centrifuged 30 minutes with 16,000g at 4 DEG C.Supernatant is allowed to reach room Temperature, and 130 μ L1%NaOH are added, so that the pH of liquid reaches 8.With vigorous agitation, 0.81mL is slowly added at room temperature Ethanol so that the concentration of alcohol in liquid reaches 20%.Liquid is kept at room temperature 30 minutes, and at room temperature with 16,000g is centrifuged 30 minutes.Supernatant is placed in 15 minutes in ice water bath, and with vigorous agitation, is slowly added to 1.3mL 1% acetic acid at 0 DEG C, so that the pH of liquid reaches 4.With vigorous agitation, ethanol of the 1.5mL at 0 DEG C is slowly added to, with The concentration of alcohol in liquid is made to reach 34%.Make liquid be kept for 30 minutes at 0 DEG C, and 30 are centrifuged with 16,000g at 4 DEG C Minute.Agglomerate is resuspended in 200 μ L DI water, and is obtained 20 μ L samples and is analyzed：Fig. 5, swimming lane 3.With DI water by residue Thing (180 μ L) is diluted to 2mL, and passes through 100kDa membrane filtrations.Retentate (150 μ L) is diluted to into 2mL with DI water, and again It is secondary by identical membrane filtration.The dilution and ultrafiltration of retentate is repeated once.Subsequently obtain the sample of retentate and pass through SDS PAGE is analyzed：Sample in Fig. 5, swimming lane 4.Permeate what its film retained by having the protein less than 100kDa molecular weight The MS2 coat proteins of 14kDa be it is clear, it is consistent with the presence of complete MS2 capsids.Show with regard to identical in Fig. 6 The UV spectrum of retentate, this with by G.F.Rohrmann and R.G.Krueger, (1970) J.Virol., 6 (3):26 for pure Result disclosed in MS2 phagies is consistent.Using with the Tris buffer saline and 150mMNaCl of pH7.4, identical retentate is run Superdex 200 (GE Healthcare, Piscataway, NJ) size exclusion chromatography (SEC).It shows the void volume only in post The 280nm absorbances at place.For the protein of 600kD to 2kD, there is no absorbance in elution volume.The test with it is complete Phage particle is consistent.Using QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) and without DNA test kits (Life Technologies, Grand Island, NY), from another sample of identical retentate separates RNA, and makes With High Capacity cDNA Reverse Transcription Kit (Life Technologies) reverse transcriptions.Subsequently In PCR experiment, three of inquiry MS2 genomes different sections is presence or absence of.Using following primer pair, every kind of primer Named in positive (F) and the reversely position of (R) first and last base in MS2 genomes by which respectively： F1001_1021-R2180_2201、F1201_1223-R1979_2001、F1401_1426-R1680_1705。Platinum Taq DNA Polymerase High Fidelity (Life Technologies) is for expanding.As shown in Figure 9 (, for primers F 1201_1223-R1979_2001 in swimming lane 1,800bp is for primers F 1201_ in swimming lane 2 for 1.2kbp 1223-R1979_2001, and 304bp is for primers F 1401_1426-R1680_1705 in swimming lane 3), contaminated with Ethidum Eremide The PCR primer that chromatograph is carried out on 1.5% agarose gel of color is consistent with complete MS2 bacteriophages genome.It is infectious to survey Examination is also run to identical retentate as follows.When bacterial culturess reach the point of OD (600nm)=0.22,5 μ L retentates are used for Infection is such as the 1mL bacterial culturess described in example A.As shown in Figure 7, OD (600nm) after infection 1 hour be 0.621, and And 0.21 was down to after other 2 hours, and at the same time, the OD (600nm) that control sample obtains 0.82 in 1 hour after infection, and And 1.2 OD (600nm) is obtained after other 2 hours.The test is displayed in high infective phage in retentate, and because This confirms not damaging its integrity for separating its purification process.In a word, the product of acquisition contains and has higher than 99% purity MS2 bacteriophages.

Example G：Using the MS2 bacteriophage purification of different exogenous proteases and ultrafiltration

Substantially as the MS2 bacteriophage purification using different exogenous proteases is attempted described in example E, except making Beyond the protease different from E.C. 3.4.21.64.By protease (P5380, the Sigma from Bacillus licheniformis Aldrich after the proteolysiss for) promoting, successful purification MS2 bacteriophages.However, using from hog gastric mucosa under pH6 The proteolysiss reaction of pepsin (P6887, Sigma Aldrich) finds significantly degraded MS2 bacteriophages.The opposing party Face, the proteolysiss reaction using the papain (P3125, Sigma Aldrich) from papaya latex under pH6 be not extensive Degraded MS2 bacteriophages.

Example H：The production of the MS2 capsids of encapsidate RNA, the RNA encode which and are attached to 19 aggressiveness RNA of its specificity The coat protein of folder

It is carried out as follows the production of MS2 capsids.Sample is obtained during expression process, and sample operation SDS PAGE are divided Analysis, to monitor capsid production.The result of acquisition is shown in Fig. 8.By the coat protein and its specific RNA 19 of coding MS2 Following DNA sequence in aggressiveness PAC sites are cloned in pDEST14_A252 plasmids (LifeTechnologies)：

ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGA CAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACT CGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTC GAGGTGCCTAAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATAT GGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAG ATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAGACGCCGGCCATTCAAACATGAGGA TTACCCATGTACCCAGCT(SEQ ID NO:6)

One Shot BL21 (DE3) Competent escherichia coli (Life are converted using such plasmid Technologies) cell.BL21 containing the plasmid (DE3) contains in 750mL in the LB culture medium of ampicillin 37 OD (600nm) is grown at DEG C equal to 0.8.Subsequently obtain pre-induction sample and analyze：Sample in Fig. 8, swimming lane 1.Subsequently Add the final concentration of isopropyl ss-D-1- Thiogalactopyranosides (Sigma-Aldrich) to 1mM.Four hours after induction, lead to Cross 40 minutes harvestings of centrifugation at 3,000g and 4 DEG C.Subsequently obtain sample and analyze：Sample in Fig. 8, swimming lane 2.

Example I：The purification and sign of the MS2 capsids of encapsidate RNA, the RNA encode its be attached to its specificity 19 gather The coat protein of body RNA hair clips

The purification of MS2 capsids is carried out as follows.Sample is obtained in purge process, and sample operation SDS PAGE are divided Analysis.The result of acquisition is shown in Fig. 8.Will be equivalent to 115mL cultures the agglomerate fraction from example H be resuspended to containing The 20mM Tris-HCl of 10mM MgCl2, in pH7.5, and supersound process is with cell lysis.By being gone with 16,000g centrifugations Except cell debriss.Such as by Strauss ＆ Sinsheimer (1963) J.Mol.Biol7:43-54 descriptions, by using sulphuric acid The precipitation of ammonium and the extraction using Arcton 11 (freon-11), the cell lysate that partial purification is obtained.To partial purification MS2 capsid solution (1.05mL) in, addition is dissolved in 0.3mg E.C. 3.4.21.64s in 1.05mL20mM CaCl2 aqueous solutions.Make to mix Compound is incubated at 37 DEG C, and after 2.5 hours, is placed in ice-water bath.Subsequently obtain sample and analyze：In Fig. 8, swimming Sample in road 3.After 15 minutes, with vigorous agitation, 0.14mL1% phosphate aqueous solutions are slowly added in ice water bath, with The pH of liquid is made to reach 4.1.Liquid is kept for 30 minutes at 0 DEG C, and be centrifuged 20 minutes with 16,000g at 4 DEG C.Xiang Bao Hold in the supernatant at 0 DEG C, add 100 μ L1%NaOH, so that the pH of liquid reaches 7.9.Subsequently with vigorous agitation, delay It is slow to add ethanol of the 0.5mL at 0 DEG C, so that the concentration of alcohol in liquid reaches 20%.Liquid is made to be kept for 30 points at 0 DEG C Clock, and be centrifuged 20 minutes with 16,000g at 4 DEG C.After adding 1% acetic acid the pH of solution to be adjusted to 7, pass through Vivaspin2 (Sartorius) 300kDa membrane filtration supernatant, and by 100kDa membrane filtration filtrates, 150 μ L are obtained by which Retentate.Retentate is diluted to into 2mL with phosphate buffered saline (PBS), and passes through identical 100kDa membrane filtrations.Retentate (150 μ L dilution and ultrafiltration) repeats four times.Subsequently obtain the sample of retentate and analyzed by SDS PAGE：In Fig. 8, swimming lane 4 Sample.MS2 coat proteins by the 14kDa of the film reservation which is permeated with the protein less than 100kDa molecular weight are bright It is really visible, it is consistent with the presence of complete MS2 capsids.Using QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) and without DNA test kits (Life Technologies, Grand Island, NY), from the another of identical retentate RNA is separated in a sample, and uses High Capacity cDNA Reverse Transcription Kit (Life Technologies) reverse transcription.Subsequently inquire about the presence or absence of of MS2 shell sections in PCR experiment.Using following primer It is right, every kind of primer by its respectively at positive (F) and reverse (R) in MS2 genomes first with the position of last base Put and name：F1401_1426-R1680_1705.Platinum Taq DNA Polymerase High Fidelity(Life Technologies) for expanding.(the 304bp in swimming lane 1 as shown in Figure 10；Leftmost swimming lane is corresponding to from Life The 1kb plus of Technologies are terraced), the PCR primer of chromatograph is carried out on 2% agarose gel dyeed with Ethidum Eremide It is consistent with complete MS2 envelope genes.In a word, the product of acquisition contains with the MS2 capsids higher than 99% purity.

Example J：It is used for purification MS2 virus-like particles (VLP) using the simple precipitation of ethanol

The purification of MS2 VLP is carried out as follows.Sample is obtained in purge process, and sample operation SDS PAGE are divided Analysis.The result of acquisition is shown in Figure 11.By derive from example H identicals experiment 1/6th agglomerates be resuspended to containing 10mM MgCl₂20mM Tris-HCl, in pH7.5, and supersound process is with cell lysis.By being gone with 16,000g centrifugations Except cell debriss.Such as by Strauss ＆ Sinsheimer (1963) J.Mol.Biol7:43-54 descriptions, by using sulphuric acid The precipitation of ammonium and the extraction using Arcton 11 (freon-11), the cell lysate that partial purification is obtained.Obtain sample simultaneously Analysis：Sample in Figure 11, swimming lane 1.It was found that the strong band at about 14kDa, the coat protein one with MS2 phagies Cause.There is great majority other bands-impurity of higher molecular weight to represent about 27% sample quality.To partially purified MS2 In VLP solution (1.35mL), 1.36mL20mM CaCl2 aqueous solutions are added, is placed in ice-water bath.After 15 minutes, 50 are added μ L10% acetic acid aqueous solutions, so that the pH of liquid reaches 4.1.Subsequently, at the same temperature and with vigorous agitation, it is slowly added to 1.44mL ethanol.Liquid is kept for 30 minutes at 0 DEG C, and be centrifuged 20 minutes with 16,000g at 4 DEG C.Agglomerate is suspended In adjusting to pH7.5 by 20mM Tris-HCl and 10mM MgCl₂In the 2mL water buffer of composition.Obtain sample and pass through SDS PAGE are analyzed：Sample in Figure 11, swimming lane 2.Impurity in the sample represents about 24% example weight.Pass through Vivaspin 2 (Sartorius) 100kDa membrane filtration dilute samples, obtain 200 μ L retentates by which.Identical buffering is used subsequently Retentate is diluted to 2mL by liquid, and passes through identical 100kDa membrane filtrations.The dilution and ultrafiltration of retentate (200 μ L) is repeated Four times.Subsequently obtain the sample of retentate and analyzed by SDS PAGE：Sample in Figure 11, swimming lane 3.It is miscellaneous in the sample Matter represents about 9.7% example weight.In a word, the product of acquisition contains with the MS2 VLP higher than 99% purity.

Example K：It is used for purification MS2VLP using E.C. 3.4.21.64 (PK) and using the simple precipitation of ethanol.

The purification of MS2 VLP is carried out as follows.Sample is obtained in purge process, and sample operation SDS PAGE are divided Analysis.The result of acquisition is shown in Figure 12.By derive from example H identicals experiment 1/6th agglomerates be resuspended to containing 10mM MgCl₂20mM Tris-HCl, in pH7.5, and supersound process is with cell lysis.By being gone with 16,000g centrifugations Except cell debriss.Such as by Strauss ＆ Sinsheimer (1963) J.Mol.Biol7:43-54 descriptions, by using sulphuric acid The precipitation of ammonium and the extraction using Arcton 11 (freon-11), the cell lysate that partial purification is obtained.Obtain sample simultaneously Analysis：Sample in Figure 12, swimming lane 1.It was found that the strong band at about 14kDa, the coat protein one with MS2 phagies Cause.There is great majority other bands-impurity of higher molecular weight to represent about 26% sample quality.To partially purified MS2 In VLP solution (1.35mL), addition is dissolved in the 0.6mg E.C. 3.4.21.64s in 1.36mL20mM CaCl2 aqueous solutions.Mixture is made to exist Incubate at 37 DEG C, and after 2.5 hours, be placed in ice-water bath.Obtain sample and analyzed by SDS PAGE：In Figure 12, swimming Sample in road 2.Impurity in the sample represents about 14% example weight.After 15 minutes, about 50 are added in ice water bath μ L10% acetic acid aqueous solutions, so that the pH of liquid reaches 4.1.Subsequently, at the same temperature and with vigorous agitation, it is slowly added to 1.54mL ethanol.Liquid is kept for 30 minutes at 0 DEG C, and be centrifuged 20 minutes with 16,000g at 4 DEG C.Agglomerate is suspended In adjusting to pH7.5 by 20mM Tris-HCl and 10mM MgCl₂In the 2mL water buffer of composition.Obtain sample and pass through SDS PAGE are analyzed：Sample in Figure 12, swimming lane 3.Impurity in the sample represents about 10% example weight.Pass through The sample of Vivaspin2 (Sartorius) 100kDa membrane filtrations dilution, obtains 200 μ L retentates by which.Identical buffering is used subsequently Retentate is diluted to 2mL by liquid, and passes through identical 100kDa membrane filtrations.The dilution and ultrafiltration of retentate (200 μ L) is repeated Four times.Subsequently obtain the sample of retentate and analyzed by SDS PAGE：Sample in Figure 12, swimming lane 4.It is miscellaneous in the sample Matter represents about 5.1% example weight.In a word, the product of acquisition contains the MS2 VLP with about 95% purity.

Example L：It is used for purification MS2VLP using composition hydrolytic enzyme (CH), with ethanol precipitation and ultrafiltration.

The purification of MS2 VLP is carried out as follows.Sample is obtained in purge process, and sample operation SDS PAGE are divided Analysis.The result of acquisition is shown in Figure 13.By derive from example H identicals experiment 1/6th agglomerates be resuspended to containing 10mM MgCl₂20mM Tris-HCl, in pH7.5, and supersound process is with cell lysis.By being gone with 16,000g centrifugations Except cell debriss.Such as by Strauss ＆ Sinsheimer (1963) J.Mol.Biol7:43-54 descriptions, by using sulphuric acid The precipitation of ammonium and the extraction using Arcton 11 (freon-11), the cell lysate that partial purification is obtained.To partial purification MS2 VLP solution (1.35mL) in, add 1.36mL20mM CaCl₂Aqueous solution.Make mixture at 37 DEG C at 2.5 hours During (with allow composition hydrolysis enzyme effect) incubate, be placed in ice-water bath thereafter.Obtain sample and by SDS PAGE point Analysis：Sample in Figure 13, swimming lane 1.Impurity in the sample represents about 12% example weight.After 15 minutes, in ice/water About 120 μ L1% sodium hydrate aqueous solutions are added in bath, so that the pH of liquid reaches 7.86.Subsequently, at the same temperature it is and adjoint Vigorous agitation, is slowly added to 0.81mL ethanol.Liquid is kept for 30 minutes at 0 DEG C, and be centrifuged with 16,000g at 4 DEG C 20 minutes.With the vigorous agitation in ice water bath, about 100 μ L10% acetic acid aqueous solutions are slowly added to in supernatant, so that The pH of liquid reaches 4.01.Subsequently, at the same temperature and with vigorous agitation, it is slowly added to 1.3mL ethanol.Liquid is made 0 Kept for 30 minutes at DEG C, and be centrifuged 20 minutes with 16,000g at 4 DEG C.Agglomerate is suspended in adjust to pH7.5 by 20mMTris-HCl and 10mMMgCl₂In the 2mL water buffer of composition.By Vivaspin2 (Sartorius) 100kDa film mistakes The sample of filter dilution, obtains 200 μ L retentates by which.Retentate is diluted to into 2mL with same buffer subsequently, and passes through phase With 100kDa membrane filtrations.The dilution and ultrafiltration of retentate (200 μ L) repeats four times.Subsequently obtain the sample of retentate and pass through SDS PAGE are analyzed：Sample in Figure 13, swimming lane 3.Impurity in the sample represents about 4.7% example weight.In a word, obtain The product for obtaining contains the MS2 VLP with greater than about 95% purity.

Example M：It is used for purification MS2VLP using E.C. 3.4.21.64 (PK), with ethanol precipitation and ultrafiltration.

The purification of MS2 VLP is carried out as follows.Sample is obtained in purge process, and sample operation SDS PAGE are divided Analysis.The result of acquisition is shown in Figure 13.By derive from example H identicals experiment 1/6th agglomerates be resuspended to containing 10mM MgCl₂20mM Tris-HCl, in pH7.5, and supersound process is with cell lysis.By being gone with 16,000g centrifugations Except cell debriss.Such as by Strauss ＆ Sinsheimer (1963) J.Mol.Biol7:43-54 descriptions, by using sulphuric acid The precipitation of ammonium and the extraction using Arcton 11 (freon-11), the cell lysate that partial purification is obtained.To partial purification MS2 VLP solution (1.35mL) in, addition be dissolved in 1.36mL20mM CaCl₂0.3mg E.C. 3.4.21.64s in aqueous solution.Make to mix Compound was incubated at 37 DEG C during 2.5 hours, was placed in ice-water bath thereafter.Obtain sample and analyzed by SDS PAGE： Sample in Figure 13, swimming lane 2.Impurity in the sample represents about 8.1% example weight.After 15 minutes, in ice water bath About 120 μ L1% sodium hydrate aqueous solutions of middle addition, so that the pH of liquid reaches 7.86.Subsequently, at the same temperature and with acute Strong agitation, is slowly added to 0.81mL ethanol.Make liquid be kept for 30 minutes at 0 DEG C, and 20 are centrifuged with 16,000g at 4 DEG C Minute.About 100 μ L10% acetic acid aqueous solutions are added in supernatant in ice water bath, so that the pH of liquid reaches 4.01.With Afterwards, at the same temperature and with vigorous agitation, it is slowly added to 1.3mL ethanol.Liquid is made to be kept for 30 minutes at 0 DEG C, and It is centrifuged 20 minutes with 16,000g at 4 DEG C.Agglomerate is suspended in and is adjusted to pH7.5 by 20mM Tris-HCl and 10mM MgCl₂In the 2mL water buffer of composition.The sample diluted by Vivaspin2 (Sartorius) 100kDa membrane filtrations, by which Obtain 200 μ L retentates.Retentate is diluted to into 2mL with same buffer subsequently, and passes through identical 100kDa membrane filtrations.Ooze The dilution and ultrafiltration of excess (200 μ L) repeats four times.Subsequently obtain the sample of retentate and analyzed by SDS PAGE：In figure 13, the sample in swimming lane 4.Impurity in the sample represents about 0.9% example weight.In a word, the product of acquisition contains and has The MS2 VLP of greater than about 99% purity.

Example N：It is used for purification MS2VLP using various hydrolytic enzyme and with the fractional precipitation of ammonium sulfate.

The purification of MS2 VLP is carried out as follows.Sample is obtained in purge process, and sample operation SDS PAGE are divided Analysis.The result of acquisition is shown in Figure 14.By derive from example H identicals experiment 1/6th agglomerates be resuspended to containing The 20mM Tris-HCl of 10mM MgCl2, in pH7.5, and supersound process is with cell lysis.By being gone with 16,000g centrifugations Except cell debriss.Obtain supernatant samples and analyzed by SDS PAGE：Sample in Figure 14, swimming lane 1.It is miscellaneous in the sample Matter represents about 70% example weight.In the same manner processing derive from four of the such experiment of example H identicals other are identical Agglomerate fraction.

The cell lysate of five centrifugations of each 3.7mL of volume of acquisition is processed with five kinds of different modes as follows.The The cell lysate of one centrifugation is placed in 15 minutes in ice-water bath, and adds 0.1 gram of ammonium sulfate.Mixture is vortexed until realizing Ammonium sulfate is completely dissolved.Liquid is kept for 2 hours at 0 DEG C, and be centrifuged 30 minutes with 16,000g at 4 DEG C.By 0.4 During gram ammonium sulfate adds supernatant, and it is vortexed until realizing being completely dissolved for ammonium sulfate.Liquid is made to be kept for 2 hours at 0 DEG C, And it is centrifuged 30 minutes with 16,000g at 4 DEG C.The MS2VLP agglomerates of purification are suspended in and are adjusted to pH 7.5 by 20mM In the 0.2mL water buffer of Tris-HCl and 10mM MgCl2 compositions.The cell lysate of the second centrifugation incubates five at 37 DEG C Hour, the cell lysate identical time quantum with the first centrifugation is placed in ice-water bath, and subsequently with thin with the first centrifugation Cellular lysate thing identical mode is processed.The 3rd is added to be centrifuged 0.15mg E.C. 3.4.21.64s (Sigma Aldrich, St.Louis, MO) Cell lysate.It is incubated five hours at 37 DEG C, be placed in ice-water bath the cell lysate phase with the first centrifugation Same time quantum, and subsequently processed in the cell lysate identical mode with the first centrifugation.Split the cell of the 4th centrifugation Solution thing is incubated two hours at 37 DEG C, is subsequently added 0.15mg PK.It is incubated at 37 DEG C other three hours, be placed in frozen water Cell lysate identical time quantum in bath with the first centrifugation, and subsequently with the cell lysate identical with the first centrifugation Mode is processed.

By 500 unitsNuclease (Sigma Aldrich, St.Louis, MO) and 35 units are from wrinkle The lipase (Sigma Aldrich, St.Louis, MO) of pleat candida mycoderma (Candida rugosa) adds the thin of the 5th centrifugation Cellular lysate thing, and incubate one hour at 37 DEG C.15 units are subsequently added from Bacillus spec (Bacillus Sp. α-amylase (Sigma Aldrich, St.Louis, MO)), and other one hour is incubated at 37 DEG C.It is subsequently added 0.15mgPK.Mixture is incubated at 37 DEG C other three hours, be placed in ice-water bath the cell lysate phase with the first centrifugation Same time quantum, and subsequently processed in the cell lysate identical mode with the first centrifugation.

The sample of the cell lysate of the second centrifugation is obtained after incubating at its 5 hours, and is analyzed by SDS PAGE：In figure 14, the sample in swimming lane 2.The sample of the cell lysate of the 3rd centrifugation is obtained after incubating at its 5 hours, and passes through SDS PAGE Analysis：Sample in Figure 14, swimming lane 3.The sample of the cell lysate of the 4th centrifugation is obtained after incubating at its 5 hours, and is led to Cross SDS PAGE analyses：Sample in Figure 14, swimming lane 4.The cell lysate of the 5th centrifugation is obtained after incubating at its 5 hours Sample, and analyzed by SDS PAGE：Sample in Figure 14, swimming lane 5.

For the sample of the MS2 VLP suspensions that the cell lysate of the first centrifugation obtains purification, and pass through SDS PAGE Analysis：Sample in Figure 14, swimming lane 6.The product of acquisition contains the MS2VLP with about 88% purity.The protein of the sample Concentration (BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, IL) be 18.5mg/mL.The 200 of the sample:The optical density (OD-260nm) that 1 dilution factor is measured in 260nm is in 1cm cells is 0.553, and OD-280nm is 0.303.These measurements are consistent with the RNA yield that about 9mg/ rises culture.

For the sample of the MS2 VLP suspensions that the cell lysate of the second centrifugation obtains purification, and pass through SDS PAGE Analysis：Sample in Figure 14, swimming lane 7.The product of acquisition contains the MS2 VLP with about 75% purity.The albumen of the sample Matter concentration is 25.4mg/mL.The 200 of the sample:Optical density (the OD- that 1 dilution factor is measured in 260nm is in 1cm cells It is 260nm) 0.784, and OD-280nm is 0.453.These measurements are consistent with the RNA yield that about 11mg/ rises culture.

For the sample of the MS2 VLP suspensions that the cell lysate of the 3rd centrifugation obtains purification, and pass through SDS PAGE Analysis：Sample in Figure 14, swimming lane 8.The product of acquisition contains the MS2 VLP with about 94.3% purity.The egg of the sample White matter concentration is 21.0mg/mL.The 200 of the sample:Optical density (the OD- that 1 dilution factor is measured in 260nm is in 1cm cells It is 260nm) 0.632, and OD-280nm is 0.321.These measurements are consistent with the RNA yield that about 10mg/ rises culture.

For the sample of the MS2 VLP suspensions that the cell lysate of the 4th centrifugation obtains purification, and pass through SDS PAGE Analysis：Sample in Figure 14, swimming lane 9.The product of acquisition contains the MS2 VLP with about 95.6% purity.The egg of the sample White matter concentration is 19.4mg/mL.The 200 of the sample:Optical density (the OD- that 1 dilution factor is measured in 260nm is in 1cm cells It is 260nm) 0.666, and OD-280nm is 0.353.These measurements are consistent with the RNA yield that about 11mg/ rises culture.

For the sample of the MS2 VLP suspensions that the cell lysate of the 5th centrifugation obtains purification, and pass through SDS PAGE Analysis：Sample in Figure 14, swimming lane 10.The product of acquisition contains the MS2 VLP with about 96% purity.The albumen of the sample Matter concentration is 19.8mg/mL.The 200 of the sample:Optical density (the OD- that 1 dilution factor is measured in 260nm is in 1cm cells It is 260nm) 0.661, and OD-280nm is 0.354.These measurements are consistent with the RNA yield that about 11mg/ rises culture.

Example O：The production of the MS2 capsids of encapsidate shRNA, the shRNA targeting green fluorescent protein (GFP) and attachment To the HDV ribozymes of MS219 aggressiveness RNA hair clips.

It is carried out as follows the production of MS2 capsids.By following DNA sequence of the coat protein of coding MS2, sequence A (SEQ ID NO:7) it is cloned in pDEST14 (Life Technologies) plasmid：

ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGA CAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACT CGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTC GAGGTGCCTAAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATAT GGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAG ATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG sequences A (SEQ ID NO:7)

By following DNA sequence, sequence B (SEQ ID NO:8) it is cloned in plasmid pACYC184.Also by transcription terminator gram It is grand to sequence B (SEQ ID NO:8) 3' ends (not shown).Sequence B (SEQ ID NO:8) encodeShRNA hair clips, design To cut hepatitis D virus (HDV) ribozyme of the 3' ends of siRNA hair clips, and by 19 aggressiveness gram of specific RNA of MS2 It is grand in plasmid pACYC184：

Sequence T7-Rz6

GGATCCTAATACGACTCACTATAGGCAAGCTGACCCTGAAGTTCTCAAGAGGAACTTCAGGGTCAGCTTGCCAAGGC CGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCACGCTTCAAACATGAGG ATTACCCATGTCGAAGCGACCATGG(SEQ ID NO:8)。

Using respectively containing sequence A (SEQ ID NO:104) with sequence B (SEQ ID NO:105) 2 kinds of plasmid conversion One Shot BL21 (DE3) Competent escherichia coli (Life Technologies) cell, selects chloromycetin and ammonia benzyl penicillium sp Plain resistant transformant.For capsid is produced, these transformants are made to contain ampicillin and chloromycetin two in 32mL at 37 DEG C Grow in the LB culture medium of person.When culture density reaches OD (600nm)=0.8, isopropyl ss-D-1- is subsequently added thio The final concentration of synthesis (Sigma-Aldrich) to 1mM.4 hours after induction, by being centrifuged at 3,000g and 4 DEG C 40 minutes harvestings.RNA is extracted as described in example Z from the VLP of purification, and as analyzed described in example AA.Such as Figure 16 In swimming lane shRNA in observe, it was observed that as encode shRNA expected from same molecular amount band.

Example P：Using the transcript production MS2 capsids of coding siGFP, flanks of the siGFP on its 5' end is Long hammerhead ribozyme, and the flank on its 3' end is the HDV ribozyme and the 2nd HDV for being attached to MS219 aggressiveness RNA hair clips Ribozyme.

By following DNA sequence, sequence C (SEQ ID NO:9) it is cloned in plasmid pACYC184.Also by transcription terminator gram It is grand to sequence C (SEQ ID NO:9) 3' ends (not shown).Sequence C (SEQ ID NO:9) T7 promoteres, design are encoded For the hammerhead ribozyme of the 5' ends of cutting siRNA hair clips, siRNA hair clips, the fourth for being designed as the 3' ends for cutting siRNA hair clips 19 aggressiveness of specific RNA of Hepatitis virus (HDV) ribozyme and MS2, and another kind of HDV ribozymes are cloned into into plasmid In pACYC184：

Sequence T7-Rz12

GGATCCTAATACGACTCACTATAGGGAGATAAATAAATAAATTTGAATGAACTTCAGGGTCAGCTTGCTGATGAGGC GCTTCGGCGCCGAAACACCCAGTGGTGTCCAAGCTGACCCTGAAGTTCATTCAAGAGATGAACTTCAGGGTCAGCTT GTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCACGCTTCAAAC ATGAGGATTACCCATGTCGAAGCGAATTTATTTATTTAATTATTATTATTATTATTGGCCGGCATGGTCCCAGCCTC CTCGCTGGCGCCGGCTGGGCAACACCTTCGGGTGGCGAATGGGACCAAAAAAAAATAATAATAATAATAATCCATGG (SEQ ID NO:9)

Using respectively containing sequence A (SEQ ID NO:104) with sequence C (SEQ ID NO:106) 2 kinds of plasmid conversion One Shot BL21 (DE3) Competent escherichia coli (Life Technologies) cell, selects chloromycetin and ammonia benzyl penicillium sp Plain resistant transformant.For capsid is produced, these transformants are made to contain ampicillin and chloromycetin two in 32mL at 37 DEG C Grow in the LB culture medium of person.When culture density reaches OD (600nm)=0.8, isopropyl ss-D-1- is subsequently added thio The final concentration of synthesis (Sigma-Aldrich) to 1mM.4 hours after induction, by being centrifuged at 3,000g and 4 DEG C 40 minutes harvestings.

Example Q：MS2 capsids are produced using transcript, the transcript encodes shRNA and is attached to 19 aggressiveness RNA of MS2 The slow HDV ribozymes of hair clip.

As carry out described in example O produce MS2 capsids several experiments, the MS2 capsids each encapsidate difference goods. However, replacing in sequence B (SEQ ID NO:8) the HDV ribozyme sequences for including, with slower anti-used in each experiment Answer the mutant of the 3' ends of speed constant cutting siRNA.For each experiment, using following containing slower HDV ribozymes Sequence replaces sequence B：

Sequence G76U：

GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCTAATGGGACCATATATATATACAT GAGGATTACCCATGTCCATGG(SEQ ID NO:10)

Sequence G40U：

GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGTCAACATTCGTGGCGAATGGGACCATATATATATACAT GAGGATTACCCATGTCCATGG(SEQ ID NO:11)

Sequence A16G：

TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCGGCCGGCATG GTCCCGGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGATTACC CATGTCCATGG(SEQ ID NO:12)

Sequence G39U：

TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCGGCCGGCATG GTCCCAGCCTCCTCGCTGGCGCCGGCTGTGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGATTACC CATGTCCATGG(SEQ ID NO:13)

Sequence A78G：

TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCGGCCGGCATG GTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAGTGGGACCATATATATATACATGAGGATTACC CATGTCCATGG(SEQ ID NO:14)

Sequence C 21U：

TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCGGCCGGCATG GTCCCAGCCTTCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGATTACC CATGTCCATGG(SEQ ID NO:15)

Sequence G25A：

TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCGGCCGGCATG GTCCCAGCCTCCTCACTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGATTACC CATGTCCATGG(SEQ ID NO:16)

Sequence A78U：

TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCGGCCGGCATG GTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGATTGGGACCATATATATATACATGAGGATTACC CATGTCCATGG(SEQ ID NO:17)

Sequence G74C：

TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCGGCCGGCATG GTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGCCGAATGGGACCATATATATATACATGAGGATTACC CATGTCCATGG(SEQ ID NO:18)

Example R：Using the transcript production MS2 capsids of coding shGFP, flanks of the shGFP on its 5' end is A kind of long hammerhead ribozyme, and the flank on its 3' end is another kind of long tup for being attached to MS219 aggressiveness RNA hair clips Shape ribozyme and HDV ribozymes.

ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTCTAACTTTACTCAGTTCGTT CTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAG CTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCA TCAAAGTCGAGGTGCCTAAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCT CCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG

By following DNA sequence, sequence D (SEQ ID NO:19) it is cloned in plasmid pACYC184.Also by transcription terminator It is cloned into sequence D (SEQ ID NO:19) 3' ends (not shown).Sequence D (SEQ ID NO:19) encode T7 promoteres, It is designed as cutting the hammerhead ribozyme of 5' ends of siRNA hair clips, siRNA hair clips, is designed as cutting the 3' ends of siRNA hair clips Hammerhead ribozyme, 19 aggressiveness of specific RNA of MS2 and HDV ribozymes：

Sequence T7-Rz15：

GGATCCTAATACGACTCACTATAGGGAGACGTTCACGTTGAATGAACTTCAGGGTCAGCTTGCTGATGAGGCGCTTC GGCGCCGAAACACCCAGTGGTGTCCAAGCTGACCCTGAAGTTCATTCAAGAGATGAACTTCAGGGTCAGCTTGTCAC CGGATGTGCTCTCCGGTCTGATGAGTCCGTGAGGACGAAACAAGCTGACCCTGAAGTTCATCCGTGAACGACGCTTC AAACATGAGGATTACCCATGTCGAAGCGAATATATATATATAGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGG CTGGGCAACACCTTCGGGTGGCGAATGGGACCAAAAAAATATATATATATACCATGG(SEQIDNO:19)

Using respectively containing sequence A (SEQ ID NO:7) with sequence D (SEQ ID NO:19) 2 kinds of plasmid conversion One Shot BL21 (DE3) Competent escherichia coli (Life Technologies) cell, selects chloromycetin and ammonia benzyl penicillium sp Plain resistant transformant.For capsid is produced, these transformants are made to contain ampicillin and chloromycetin two in 750mL at 37 DEG C Grow in the LB culture medium of person.When culture density reaches OD (600nm)=0.8, isopropyl ss-D-1- is subsequently added thio The final concentration of synthesis (Sigma-Aldrich) to 1mM.4 hours after induction, by being centrifuged at 3,000g and 4 DEG C 40 minutes harvestings.Sample is obtained before induction and when harvesting to be used to analyze.

Example S：MS2 capsids are produced using transcript, the transcript encodes shRNA and is attached to 19 aggressiveness RNA of MS2 The long hammerhead ribozyme of hair clip

As carry out described in example O produce MS2 capsids several experiments, the MS2 capsids each encapsidate difference goods. However, replacing in sequence B (SEQ ID NO:8) the HDV ribozyme sequences for including, the long hammerhead ribozyme used in each experiment Sequence, which hybridizes and is designed as cutting which with the 3' ends of siRNA.For each experiment, use containing long hammerhead ribozyme Following sequences replace sequence B (SEQ ID NO:8)：

Sequence 10MNT (not good to cut)：

ATATATATATACATGAGGATTACC CATGTCCATGG (SEQ ID NO:20)

Sequence 15MNT (not good to cut)：

ATATATATAT CCATGG(SEQ ID NO:21)

Sequence 20MNT (good to cut)：

ATATATATAT CCATGG(SEQ ID NO:22)

Sequence 22MNT (good to cut)：

ATATATATAT CCATGG(SEQ ID NO:23)

Sequence 25MNT (good to cut)：

ATATATATA TCCATGG(SEQ ID NO:24)

Sequence 27MNT (good to cut)：

ATATATA TATCCATGG(SEQ ID NO:25)

Experimental data described in the example supports following conclusions：According to present disclosure, well the RNA chains of cutting are to wrap The RNA chains of hammerhead ribozyme sequence are included, for the hammerhead ribozyme sequence, the appropriate folding kinetics of ribozyme sequence favorably surpass Cross the folding of shRNA (" the hair clip ") section of whole piece chain.The respective thermodynamic parameter of above-mentioned sequence is calculated, so which sequence determined Well cut, and which sequence is not.

Example T：Using the transcript production MS2 capsids of coding siRNA, flanks of the siRNA on its 5' end is Long hammerhead ribozyme, and the flank in its 3' end is the HDV cores of the trans-acting for being attached to MS219 aggressiveness RNA hair clips Enzyme.

It is carried out as follows the production of MS2 capsids.By following DNA sequence (sequences A of the coat protein of coding MS2；SEQ ID NO:7) it is cloned in pDEST14 (Life Technologies) plasmid：

ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGA CAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACT CGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTC GAGGTGCCTAAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATAT GGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAG ATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG(SEQ ID NO:7)

By following DNA sequence, sequence E (SEQ ID NO:26) it is cloned in plasmid pACYC184.Also by transcription terminator It is cloned into sequence E (SEQ ID NO:26) 3' ends (not shown).Sequence E (SEQ ID NO:26) encode BamHI to limit Site, T7 promoteres and homing sequence, it is designed as the siRNA to enhanced green fluorescence protein with reflectron mode cutting needle (siEGPF) the HDV ribozymes of 3' ends, ATAT septs, the long hammerhead shape core of the 5' ends for being designed as cutting siEGFP hair clips Enzyme, siEGFP hair clips, the complementary seriess of HDV ribozymes of 3' ends that siEGFP hair clips are cut with reflectron mode, the specificity of MS2 RNA19 aggressiveness and PacI restriction sites：

GGATCCTAATACGACTCACTATAGGGATCTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGG GGGATCATATCTTGATGAACTTCAGGGTCAGCTTGCTGATGAGGCGCTTCGGCGCCGAAACACCGTGTCCAAGCTGA CCCTGAAGTTCATCAAGAATGAACTTCAGGGTCAGCTTGTCGGCCGGCATGCATTCAAACATGAGGATTACCCATGT CGAAGTTAATTAA(SEQ ID NO:26)

BamHI and HindIII sites are added in 5' ends and 3' ends respectively, to promote to be cloned in pACYC184 (not shown).

Using respectively containing sequence A (SEQ ID NO:104) with sequence E (SEQ ID NO:26) 2 kinds of plasmid conversion One Shot BL21 (DE3) Competent escherichia coli (Life Technologies) cell, selects chloromycetin and ammonia benzyl penicillium sp Plain resistant transformant.For capsid is produced, these transformants are made to contain ampicillin and chloromycetin two in 750mL at 37 DEG C Grow in the LB culture medium of person.When culture density reaches OD (600nm)=0.8, isopropyl ss-D-1- is subsequently added thio The final concentration of synthesis (Sigma-Aldrich) to 1mM.4 hours after induction, by being centrifuged at 3,000g and 4 DEG C 40 minutes harvestings.Sample is obtained before induction and when harvesting to be used to analyze.

Example U：Using the transcript production MS2 capsids of 3 kinds of difference siRNA of coding targeting GFP, the side of the siRNA The wing is the long hammerhead ribozyme for being attached to 19 aggressiveness RNA hair clips of MS2.

By following DNA sequence, sequence F (SEQ ID NO:27) it is cloned in plasmid pACYC184.Also by transcription terminator It is cloned into sequence F (SEQ ID NO:27) 3' ends (not shown).Sequence F (SEQ ID NO:27) encode T7 promoteres and Homing sequence, it is designed as 3 kind hammerhead shape cores of the cutting needle to the 5' ends of the siRNA (siEGPF) of enhanced green fluorescence protein Enzyme, 3 kinds of different siEGFP hair clips, 3 kinds of long hammerhead ribozymes of the 3' ends for being designed as cutting siEGFP hair clips, the spy of MS2 Different in nature RNA19 aggressiveness：

GGATCCTAATACGACTCACTATAGGGAGACTTGATGAACTTCAGGGTCAGCTTGCTGATGAGGCGCTTCGGCGCCGA AACACCCAGTGGTGTCCAAGCTGACCCTGAAGTTCATCAAGAATGAACTTCAGGGTCAGCTTGTCACCGGATGTGCT CTCCGGTCTGATGAGTCCGTGAGGACGAAACAAGCTGACCCTGAAGTTCATTATATCTTGGCAGATGAACTTCAGGG TCAGCTGATGAGACTCTTCGGAGTCGAAACACCCAGTGGTGTCCTGACCCTGAAGTTCATCTGCCAAGAGCAGATGA ACTTCAGGGTCAGTCACCGGATGTGCTCTCCGGTCTGATGAGTCCGTGAGGACGAAACTGACCCTGAAGTTCATCTG CTATATCTTGTGGTGCAGATGAACTTCAGGGCTGATGAGGCTCTTCGGAGCCGAAACACCCAGTGGTGTCCCCTGAA GTTCATCTGCACCACAAGATGGTGCAGATGAACTTCAGGGTCACCGGATGTGCTCTCCGGTCTGATGAGTCCGTGAG GACGAAACCCTGAAGTTCATCTGCACCATACGCCGGCCATTCAAACATGAGGATTACCCATGTCGAAGTTAATTAA (SEQ ID NO:27)

Using respectively containing sequence A (SEQ ID NO:And other sequences F (SEQ ID NO 7):27) 2 kinds of plasmid conversion One Shot BL21 (DE3) Competent escherichia coli (Life Technologies) cell, selects chloromycetin and ammonia benzyl penicillium sp Plain resistant transformant.For capsid is produced, these transformants are made to contain ampicillin and chloromycetin two in 750mL at 37 DEG C Grow in the LB culture medium of person.When culture density reaches OD (600nm)=0.8, isopropyl ss-D-1- is subsequently added thio The final concentration of synthesis (Sigma-Aldrich) to 1mM.4 hours after induction, by being centrifuged at 3,000g and 4 DEG C 40 minutes harvestings.Sample is obtained before induction and when harvesting to be used to analyze.

Example V：Using the transcript production MS2 capsids of 3 kinds of difference siRNA of coding targeting GFP, the side of the siRNA The wing be positioned at its 5' end sept and be attached to MS219 aggressiveness RNA hair clips, positioned at the HDV ribozymes of its 3' end.

ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGA CAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACT CGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTC GAGGTGCCTAAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATAT GGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAG ATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG(SEQ ID NO7)

By following DNA sequence, sequence G (SEQ ID NO:28) it is cloned in plasmid pACYC184.Also by transcription terminator It is cloned into sequence G (SEQ ID NO:28) 3' ends (not shown).Sequence G (SEQ ID NO:28) encode BamHI to limit Site, T7 promoteres and homing sequence, for enhanced green fluorescence protein siRNA (siEGPF) 5' ends 3 Individual sept, 3 kinds of different siEGFP hair clips, 3 kinds of HDV ribozymes of the 3' ends for being designed as cutting siEGFP hair clips, the spy of MS2 Different in nature RNA19 aggressiveness and PacI restriction sites：

GGATCCTAATACGACTCACTATAGGGAGAATATATATACAAGCTGACCCTGAAGTTCATCAAGAATGAACTTCAGGG TCAGCTTGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCAATT AATTACTGACCCTGAAGTTCATCTGCCAAGAGCAGATGAACTTCAGGGTCAGTCGGCCGGCATGGTCCCAGCCTCCT CGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCAATAATAATCCCTGAAGTTCATCTGCACCACAAGATG GTGCAGATGAACTTCAGGGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAA TGGGACCCATTCAAACATGAGGATTACCCATGTCGAAGTTAATTAA(SEQ ID NO:28)

Using respectively containing sequence A (SEQ ID NO:7) with sequence G (SEQ ID NO:28) 2 kinds of plasmid conversion One Shot BL21 (DE3) Competent escherichia coli (Life Technologies) cell, selects chloromycetin and ammonia benzyl penicillium sp Plain resistant transformant.For capsid is produced, these transformants are made to contain ampicillin and chloromycetin two in 750mL at 37 DEG C Grow in the LB culture medium of person.When culture density reaches OD (600nm)=0.8, isopropyl ss-D-1- is subsequently added thio The final concentration of synthesis (Sigma-Aldrich) to 1mM.4 hours after induction, by being centrifuged at 3,000g and 4 DEG C 40 minutes harvestings.Sample is obtained before induction and when harvesting to be used to analyze.

Example W：Using the transcript production MS2 capsids of two siRNA chains of coding targeting GFP, the siRNA chains are each Flank be positioned at its 3' end long hammerhead ribozyme and be attached to MS219 aggressiveness RNA hair clips, positioned at its 5' end HDV ribozymes.

By following DNA sequence, sequence H (SEQ ID NO:29) it is cloned in plasmid pACYC184.Also by transcription terminator It is cloned into the 3' ends (not shown) of sequence H.

Sequence T7-Rz8

GGATCCTAATACGACTCACTATAGGGAGAATGAACTTCAGGGTCAGCTTGCTGATGAGGCGCTTCGGCGCCGAAACA CCGTGTCCAAGCTGACCCTGAAGTTCATGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCG TGGCGAATGGGACCATTAGCCAAGCTGACCCTGAAGTTCATCTGATGAGACTCCGAATTCGGAGTCGAAACACGGTA ACCGTGTCATGAACTTCAGGGTCAGCTTGGCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACAT TCGTGGCGAATGGGACCCATTCAAACATGAGGATTACCCATGTCGAAGCCATGG(SEQ ID NO:29)

Using respectively containing sequence A (SEQ ID NO:104) with sequence H (SEQ ID NO:126) 2 kinds of plasmid conversion One Shot BL21 (DE3) Competent escherichia coli (Life Technologies) cell, selects chloromycetin and ammonia benzyl penicillium sp Plain resistant transformant.For capsid is produced, these transformants are made to contain ampicillin and chloromycetin two in 750mL at 37 DEG C Grow in the LB culture medium of person.When culture density reaches OD (600nm)=0.8, isopropyl ss-D-1- is subsequently added thio The final concentration of synthesis (Sigma-Aldrich) to 1mM.4 hours after induction, by being centrifuged at 3,000g and 4 DEG C 40 minutes harvestings.

Instance X：Using the transcript production MS2 capsids of 2 kinds of difference siRNA of coding targeting green fluorescent protein (GFP), The flank of the siRNA is positioned at its 3' end and 5' ends, is attached to the sept of MS219 aggressiveness RNA hair clips.

By following DNA sequence, sequence I (SEQ ID NO:30) it is cloned in plasmid pACYC184.Also by transcription terminator It is cloned into sequence I (SEQ ID NO:30) 3' ends (not shown).Sequence I (SEQ ID NO:30) encode T7 promoteres and Homing sequence, Separated pin 3 septs, 2 kinds of different siEGFP to the end of the siRNA (siGPF) of green fluorescent protein 19 aggressiveness of specific RNA of hair clip, MS2：

GGATCCTAATACGACTCACTATAGGGAGAAATAATAATCAAGCTGACCCTGAAGTTCATCAAGAATGAACTTCAGGG TCAGCTTGTCAATAATAATCCGCTACCCCGACCACATGAACAAGATTCATGTGGTCGGGGTAGCGGTCAATAATAAT ACGCTTCAAACATGAGGATTACCCATGTCGAAGCGACCATGG(SEQ ID NO:30)

Using respectively containing sequence A (SEQ ID NO:7) with sequence I (SEQ ID NO:30) 2 kinds of plasmid conversion One Shot BL21 (DE3) Competent escherichia coli (Life Technologies) cell, selects chloromycetin and ammonia benzyl penicillium sp Plain resistant transformant.For capsid is produced, these transformants are made to contain ampicillin and chloromycetin two in 750mL at 37 DEG C Grow in the LB culture medium of person.When culture density reaches OD (600nm)=0.8, isopropyl ss-D-1- is subsequently added thio The final concentration of synthesis (Sigma-Aldrich) to 1mM.4 hours after induction, by being centrifuged at 3,000g and 4 DEG C 40 minutes harvestings.Sample is obtained before induction and when harvesting to be used to analyze.

Instance Y：The purification of the MS2VLP obtained in example O to X.

Using the MS2 capsids obtained in the operation purification example O to X summarized in example N.

Example Z：The separation of the RNA of encapsidate in the MS2 capsids obtained in example N

According to the scheme provided by manufacturer (Life Technologies, Grand Island, NY), useReagent, extracts the RNA of encapsidate in the MS2 capsids such as purification described in example N from each experiment.Obtain RNA by 95 DEG C, in Methanamide, heating carries out degeneration for 5 minutes, and by 17.6cmx38cmx0.04cm (W, L, T) in gel, electrophoresis is analyzed, and the gel is by 8% polyacrylamide, 8 mole of urea, 1.08%Tris alkali, 0.55% boron Acid and 0.093%EDTA are constituted.Electrophoretic buffer with the Tris alkali of gel same concentrations, boric acid and EDTA.Power is with about 40W is delivered.Using in the aqueous mixture for containing 25% Methanamide, 19% isopropanol and 15mM Tris with pH8 0.025% solution of Stains-All dyestuffs (Sigma-Aldrich, St.Louis, MO) will be gel-colored.The result of acquisition shows It is shown in Figure 15.Swimming lane numbering in Figure 15 for RNA electrophoresis refers to the identical swimming lane numbering in Figure 14 for protein electrophorese. Single RNA bands can be observed in each swimming lane, it is consistent with high-purity RNA for reclaiming in each case.

Example AA：HDV ribozymes produce shRNA in transcription in vitro.

Construct T7-Rz2 is used in vitro transcription.The construct is cloned into into pACYC184 plasmids (New England Biolabs in).One Shot BL21 (DE3) Competent escherichia coli (Life are converted using the plasmid Technologies) cell.BL21 containing the plasmid (DE3) is raw in the LB culture medium containing ampicillin at 37 DEG C Length is to OD (600nm) equal to 0.8.According to the description of manufacturer, useSpin Miniprep Kit (Qiagen) separation quality grain.NcoI (New England Biolabs) is for cutting at the internal restriction site of the structure introducing Cut detached plasmid.After digestion, template carries out purification by the electrophoresis on 1.5% agarose gel, and according to manufacturer Description, using PureLink^TMQuick Gel Extraction Kit (Life Technologies) is separated.Root According to the description of manufacturer, useT7 test kits complete reverse transcription.What RNA products were run at 70 DEG CElectrophoresis is carried out in 15% polyacrylamide TBE- urea gels (Life Technologies) of degeneration.Using bromination Second pyridine (Sigma-Aldrich) manifests RNA bands.Gel imaging is completed using Image Lab4.0.1 softwares (Bio-Rad).

Template T7-Rz2 encode as follows T7 promoteres, shRNA hair clips, be designed as cut shRNA hair clips 3' ends HDV Ribozyme, ATATATATAT septs and NcoI：

TAATACGACTCACTATAGGCTTGTGATGCTTCAGCCAAATCAAGAGTTTGGCTGAAGCATCACAAGCGGCCGGCATG GTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGATTACC CATGTCCATGG(SEQ ID NO:31)

As a result show that HDV ribozymes cut.Top strap is uncut transcript, and two relatively low bands are that expection is cut The RNA small pieces for cutting.

Gel in Figure 16 is displayed in one group of RNA labelling in Far Left swimming lane, afterwards 49 nucleotide in swimming lane ShRNA labellings, in the 3rd swimming lane at 37 DEG C run 1 hour T7-Rz2 constructs in vitro transcription and cutting, Yi Ji 4th, the body of the T7-Rz2 constructs of other a hour is run 1 hour and incubates at 42 DEG C in rightmost swimming lane at 37 DEG C Outer transcription and cutting.

In three most strong bands it is most slow with 37 higher than the intensity at 37/42.In addition, two more rapidly Band with 37/42 higher than the intensity at 37.Lowest molecular weight band in last swimming lane runs slightly 49nt shRNA reference materials are slower than, in for T7Rz2 expected from 51 nucleotide shRNA of gained.In a word, as shown in Figure 16 Result it is consistent with the hypothesis that HDV ribozymes are cut in T7-Rz2.

Example BB：Long flank hammerhead ribozyme is cut in transcription than short side wing hammerhead ribozyme significantly more in vitro High degree

Construct T7-Rz1 and T7-Rz4 are used in vitro transcription.T7-Rz1 includes common ribozyme, i.e., hybridize with less than 6 pairs The hybridization of nucleotide is cut the stem of siRNA.Flanking ribozymes of the T7-Rz4 comprising the stem with length length, the stem hybridization are cut The siRNA for cutting, i.e., with the stem more than 6 pairs of hybridising nucleotides.

Construct T7-Rz1 coding T7 promoteres, being designed as with 5 nucleotide complementary with siRNA cut siRNA Hammerhead shape (HH) ribozyme of the 5' ends of hair clip, siRNA hair clips, being designed as with 5 nucleotide complementary with siRNA are cut The HH ribozymes and NcoI restriction sites of the 3' ends of siRNA hair clips：

TAATACGACTCACTATAGGCTCGAGCAAGCCTGATGAGGCGCTTCGGCGCCGAAACACCGTGTCGCTTGTGATGCTT CAGCCAAATCAAGAGTTTGGCTGAAGCATCACAAGCTCACCGGATGTGCTCTCCGGTCTGATGAGTCCGTGAGGACG AAAGCTTGCCATGG(SEQ ID NO:32)

Construct T7-Rz4 encodes siRNA, and the flank of the siRNA is with 12 nucleotide hybridized with siRNA, is set Be calculated as cut its 5' end HH ribozymes, and with 23 nucleotide hybridized with siRNA, be designed as cut its 3' end HH Ribozyme：

TAATACGACTCACTATAGGGAGAACGCCGGCCATTCAAATAGTAAATAATAGAGGGTCAGCTTGCTGATGAGGCGCT TCGGCGCCGAAACACCGTGTCCAAGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCACCGGAT GTGCTCTCCGGTCTGATGAGTCCGTGAGGACGAAACAAGCTGACCCTGAAGTTCACTACGCCGGCCATTCAAACATG AGGATTACCCATGTCCATGG(SEQ ID NO:33)

In vitro transcription and analysis by instance Y those it is similar in the way of with these structure running body.As in Figure 17 Shown, the result obtained with construct T7-Rz1 is consistent with the hypothesis that ribozyme is cut to very less extent.Structure is used shown in Figure 17 The result for building body T7-Rz4 acquisitions is consistent with the hypothesis that ribozyme is cut to significance degree.

Example CC：Using coding for the shRNA of EGFP transcript production MS2 capsids, the flank of the shRNA be The long hammerhead ribozyme of its 5' end and another kind of long hammerhead shape of MS219 aggressiveness RNA hair clips is attached in its 3' end Ribozyme.

Following DNA sequence (construct T7-Rz4) are cloned in plasmid pACYC184.Also transcription terminator is cloned into The 3' ends (not shown) of construct T7-Rz4.

Using respectively containing sequence A (SEQ ID NO:7) with construct T7-Rz4 (SEQ ID NO:33) 2 kinds of plasmid conversions One Shot BL21 (DE3) Competent escherichia coli (Life Technologies) cells, select chloromycetin and ammonia benzyl Penicillin resistance transformant.For capsid is produced, make that these transformants contain ampicillin in 750mL at 37 DEG C and chlorine is mould Grow in the LB culture medium of both elements.When culture density reaches OD (600nm)=0.8, isopropyl ss-D-1- is subsequently added The final concentration of Thiogalactopyranoside (Sigma-Aldrich) to 1mM.4 hours after induction, by 3,000g and 4 DEG C 40 minutes harvestings of centrifugation.Sample is obtained before induction and when harvesting to be used to analyze.

Example DD：The purification of the virus-like particle obtained in example CC.

The purification of the virus-like particle produced in as carried out example CC in example N.

Example EE：The separation of the RNA in the virus-like particle obtained in example DD

According to the scheme provided by manufacturer (Life Technologies, Grand Island, NY), useReagent, extracts such as the RNA of encapsidate in the virus-like particle of purification described in example DD.The RNA of acquisition by At 95 DEG C, in Methanamide, heating carries out degeneration for 5 minutes, and by running at 70 DEG CDegeneration 15% poly- third In acrylamide TBE- urea gel (Life Technologies), electrophoresis is analyzed.Using 0.5 μ g Ethidum Eremide (Sigma- Aldrich, St.Louis, MO)/mL aqueous solutions manifest RNA bands.The result of acquisition is shown in Figure 18, in swimming lane 3.Swimming lane 1 shows Show a component substandard thing.Swimming lane 2 shows the shRNA of 49 nucleotide of length of chemosynthesis.

Example FF：The virus-like particle comprising MS2 capsids obtained in example DD is to from white side tooth mycete (Engyodontium album), the E.C. 3.4.21.64 of Bacillus licheniformis (licheniformis), from the stomach egg of hog gastric mucosa White enzyme, and be resistance from the papain of papaya latex.

Virus-like particle comprising 250mL cultures are derived from and such as the MS2 capsids of purification described in example DD be suspended in The 400 μ L20mM CaCl of pH=7.5₂In aqueous solution.

By the aliquot of the 66 μ L suspensions to pH=7.5 20mM CaCl₂Aqueous solution is diluted to 0.25mL, and And incubate at 37 DEG C.Incubate 1 hour and 4 hours after obtain sample for protein concentration (BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, IL) and SDS PAGE analyses.In this 2 parts of samples Protein concentration is respectively 3086 and 4656mg/L.SDS PAGE are shown in Figure 19, in swimming lane 1B and 6.Will be identical The protein of amount is loaded in each swimming lane (4 μ g).The group experiment is as negative control.

By 2 μ g from streptomyces griseuses protease (Sigma Aldrich, St.Louis, MO) to pH=7.5's 20mMCaCl₂Aqueous solution is diluted to 0.25mL, and incubates at 37 DEG C.Sample is obtained after incubating 1 hour and 4 hours to be used for Protein concentration and SDS PAGE analyses.Protein concentration in this 2 parts of samples is respectively 361 and 324mg/L.SDS PAGE Figure 19 is shown in, in swimming lane 1 and 7.Same amount of protein is loaded into in each swimming lane (4 μ g).The group is tested As another kind of negative control.

By 2 μ g from streptomyces griseuses protease add comprising MS2 capsids virus-like particle suspension another In 66 μ L aliquots, to the 20mM CaCl of pH=7.5₂Aqueous solution is diluted to 0.25mL, and incubates at 37 DEG C. Sample is obtained after incubating 1 hour and 4 hours is used for protein concentration and SDS PAGE analyses.Protein in this 2 parts of samples Concentration is respectively 2940 and 3012mg/L.SDS PAGE are shown in Figure 19, in swimming lane 2 and 8.By same amount of albumen Matter is loaded in each swimming lane (4 μ g).The group is tested for testing the MS2 capsids for forming virus-like particle for from Lycoperdon polymorphum Vitt chain The proteolysiss stability of the protease of mycete.It was observed that less than 10% degraded.

To the 20mM CaCl of pH=7.5₂Aqueous solution is diluted to 0.25mL, the virus-like particle comprising MS2 capsids and suspends Another 66 μ L aliquot of liquid is carried out and is heated to 95 DEG C 10 minutes and in three circulations of wet cooled on ice 10 minutes, with Realize the dismounting of virus-like particle.Subsequently 2 μ g are added in the suspension from the protease of streptomyces griseuses, and at 37 DEG C Lower incubation.Sample is obtained after incubating 1 hour and 4 hours is used for protein concentration and SDS PAGE analyses.In this 2 parts of samples Protein concentration be respectively 2601 and 3033mg/L.SDS PAGE are shown in Figure 19, in swimming lane 3 and 9.Will be identical The protein of amount is loaded in each swimming lane (4 μ g).The granule of dismounting is by the proteasome degradation from streptomyces griseuses to notable Degree.The group experiment is as positive control.

The 0.002mL20mM CaCl with pH=7.5 will be dissolved in₂Albumen of the 2 μ g in aqueous solution from streptomyces griseuses During enzyme adds 0.248mL bacteria cell cracking things, the bacteria cell cracking thing derives from the 41mL cell culture from example CC Thing, and incubate at 37 DEG C.Sample is obtained after incubating 1 hour and 4 hours is used for protein concentration and SDS PAGE analyses. Protein concentration in this 2 parts of samples is respectively 3192 and 4837mg/L.SDS PAGE are shown in Figure 19, swimming lane In 4 and 10.Last swimming lane for being labeled as Figure 19 of L shows undressed bacteria cell cracking thing.By same amount of egg White matter is loaded in each swimming lane (4 μ g).The protein in addition to MS2 capsid protein matter more than 90% is by from Lycoperdon polymorphum Vitt strepto- The proteasome degradation of bacterium.The group experiment is as another kind of positive control.

Five experiments of the group confirm the MS2 capsids of the virus-like particle for forming present disclosure to by from streptomyces griseuses The proteolysiss of protease be resistance.

By 2 μ g from Bacillus licheniformis protease (Sigma Aldrich, St.Louis, MO) to pH=7.5's 10mM sodium acetates and 5mM calcium acetate aqueous solutions are diluted to 0.25mL, and incubate at 37 DEG C.After incubating 1 hour and 4 hours Obtaining sample is used for protein concentration and SDS PAGE analyses.Protein concentration in this 2 parts of samples is respectively 976 Hes 1003mg/L.SDS PAGE are shown in Figure 19, in swimming lane 2B and 7B.Same amount of protein is loaded into into each swimming In road (4 μ g).The group experiment is as another kind of negative control.

2 μ g are added into the another of the virus-like particle suspension comprising MS2 capsids from the protease of Bacillus licheniformis In individual 66 μ L aliquots, 0.25mL is diluted to the 10mM sodium acetates and 5mM calcium acetate aqueous solutions of pH=7.5, and Incubate at 37 DEG C.Sample is obtained after incubating 1 hour and 4 hours is used for protein concentration and SDS PAGE analyses.In this 2 parts of samples Protein concentration in product is respectively 3144 and 3727mg/L.SDS PAGE are shown in Figure 19, in swimming lane 3B and 8B. Same amount of protein is loaded into in each swimming lane (4 μ g).The group tests the MS2 capsids that virus-like particle is formed for test For the proteolysiss stability of the protease from Bacillus licheniformis.It was observed that less than 10% degraded

0.25mL, the disease comprising MS2 capsids are diluted to the 10mM sodium acetates and 5mM calcium acetate aqueous solutions of pH=7.5 Another 66 μ L aliquot of malicious sample particle suspension liquid is carried out and is heated to 95 DEG C 10 minutes and in wet cooled on ice 10 minutes Three circulation, to realize the dismounting of virus-like particle.2 μ g are added into the suspension from the protease of Bacillus licheniformis subsequently In liquid, and incubate at 37 DEG C.Sample is obtained after incubating 1 hour and 4 hours is used for protein concentration and SDS PAGE point Analysis.Protein concentration in this 2 parts of samples is respectively 1769 and 1785mg/L.SDS PAGE are shown in Figure 19, In swimming lane 4B and 9B.Same amount of protein is loaded into in each swimming lane (4 μ g).The granule of dismounting is by from lichens spore bar The proteasome degradation of bacterium.The group experiment is as positive control.

To be dissolved in 2 μ g in the 0.002mL 10mM sodium acetates of pH=7.5 and 5mM calcium acetate aqueous solutions from lichens During the protease of bacillus cereuss adds 0.248mL bacteria cell cracking things, the bacteria cell cracking thing is derived from from example CC 41mL cell cultures, and at 37 DEG C incubate.Sample is obtained after incubating 1 hour and 4 hours is used for protein concentration Analyze with SDS PAGE.Protein concentration in this 2 parts of samples is respectively 3696 and 4078mg/L.SDS PAGE analysis difference Figure 19 is shown in, in swimming lane 6B and 10B.Last swimming lane for being labeled as Figure 19 of L shows that undressed bacterial cell splits Solution thing.Same amount of protein is loaded into in each swimming lane (4 μ g).The egg in addition to MS2 capsid protein matter more than 90% White matter is by the proteasome degradation from Bacillus licheniformis.The group experiment is as another kind of positive control.

Four experiments of the group confirm the MS2 capsids of the virus-like particle for forming present disclosure to by from lichens spore bar The proteolysiss of the protease of bacterium are resistances.

Three groups of other equivalent experiments confirm the MS2 capsids of the virus-like particle to form present disclosure to by following three kinds The proteolysiss of any one in protease are resistances：E.C. 3.4.21.64 from white side tooth mycete, from the stomach of hog gastric mucosa Protease (CAS numberings 9001-75-6), and from papain (CAS numberings the 9001-73-4) (Sigma- of papaya latex Aldrich,St.Louis,MO).Every kind of protease is used according to the description of manufacturer.E.C. 3.4.21.64 is used under pH=7.5, Pepsin is used under pH=1.6, and papain is used under pH=6.6.

Example GG：Capsid coat protein variant

MS2 viral capsid proteins matter (SEQ.ID No.3) is with single folded domain, and belongs to superfamily d.85 Fold family d.85.1 (RNA bacteriophage capsid protein matter), the superfamily d.85 include Leviviridae and Alloleviviridae capsid protein matter.Each capsid monomer in the family by 6 chain β lamellas subsequently for two spirals (sometimes It is described as the long spire with knot) composition.The non-covalent assembling of 180 monomers, to form (approximately spherical) disease of icosahedron Malicious capsid, wherein continuous β lamellas are towards inside capsid, and alpha-helix outside the capsid on.With regard to being d.85.1 family's light Enterobacteria phage MS2, GA (UniProt sequence identifier P07234) and FR (UniProt sequences of sliding Viraceae coat protein Column identifier P03614) viral capsid and (C-terminal of one of MS2 has been fused to another by MS2 dimers N-terminal) the MS2 capsids that formed, x-ray crystal structure has been resolved and has been placed in public domain.With regard to the egg of these structures White matter database identifier is respectively 1AQ3 (SEQ ID NO:34)、1GAV(SEQ ID NO:35)、1FRS(SEQ ID NO: 36) with 2VTU (SEQ ID NO:, and these comparison is shown in Figure 20 37).It is in all comparisons described herein, residual Base numbering is sequential residue numbering, and such as SEQ ID3 are with 0 beginning of guiding Met (M) residue that removed by cell, such as most of PDB structures are used.

MS2 viral capsid proteins matter is identical with 87% relative to the sequence respectively 59% of GA with FR viral capsid proteins matter 's.Only 56% sequence location has identical sequence, and when all three sequence considers together, the topology for main chain Learn location overlap of equal value.Root-mean-square of the MS2 viral capsid proteins matter relative to the Conformation of the main chain of GA and FR viral capsid monomers (rms) deviation is under 1A.1AQ3 monomers A is 0.89 angstrom relative to the main chain root-mean-square-deviation of 1GAV monomers 0.1AQ3 monomer A phases Main chain root-mean-square-deviation for 1FRS monomer A is 0.37 angstrom.Using freeware practicality jFATCAT rigidity (Prlic, et al., Bioinformatics26,2983-2985(2010)；www.rcsb.org/pdb/workbench/workbench.do； Www.rcsb.org/pdb/workbench/workbench.do) make comparisons, the freeware is in its standard protein In structure tool workbench at the RCSB Protein Data Banks website obtained by work familiar to structural research protein practitioner Tool.The overall folded of these protein is identical.There is no insertion or lack.In the asymmetric unit of independent refining crystallization Every kind of protein.1 angstrom of the general main chain root-mean-square-deviation of same protein or bigger on the different composition in asymmetric unit, Although the topology equivalence C alpha atoms of core tend to difference is less than about 0.45 angstrom of (Cyrus Chothia ＆ Arthur M Lesk (1986)EMBO J5,823-826).For example 1AQ3 monomers A and 1AQ3 monomer B have the root-mean-square-deviation (jFATCAT of 1.72A Rigidity), mainly due to the conformational difference in Lys66-Trp82 regions.

If identifying the enough members for folding family, family seldom or from conserved residues in unmutated sequence, The clear picture of topology Equivalent residues position occurs.Non-conservative position can be expected from a sequence to be mutated to another, and not Upset family to fold, may combine with the coordination of the spatial neighbors of one or more in folding mutation, particularly when side chain pin One or more side chain of spatial neighbors is compacted.Conserved residues are for the folding stability of protein, function or processing for example Proteolytic digestion can be crucial.Some can be consistent conservative.GenBank(Dennis A.Benson,Ilene Karsch-Mizrachi, David J.Lipman, James Ostell and David L.Wheeler (2005) Nucleic Acids Res 33, D34-D38) 353 kinds of Leviviridae coat protein sequences are accommodated at present.Deck watch shown in Figure 21 The multiple alignment of 40 kinds of complete Leviviridae coat protein sequences is shown, the coat protein sequence is from overall protein matter Sequence library UniProt (Universal Protein Resource, (The UniProt Consortium, Reorganizing the protein space at the Universal Protein Resource(UniProt)NucleicAcidsRes.40：D71-D75(2012)),http://www.uniprot.org) (referring to table 1 below) middle retrieval, and And compared with BLAST (threshold value=10, automatic Weighting Matrices column selection, without filtration, it is allowed to breach).It is all in addition to ef108465 Sequence derives from UniProt.Ef108465 is from GenBank (www.ncbi.nlm.nih.gov/genbank).In deck watch In, asterisk (*) indicates conserved residues, and x is calculated as based on the constraint of side chain solvent accessibility, hydrogen bonding demand and Conformation of the main chain can Displacement.57 (57) individual residues in the sequence of these family members are conservative, or 45% sequence is mutually the same.This Some in a little sequences are with SEQ ID NO:Other residue after 3 C-terminal Tyr129 residues, other are with regard to SEQ ID NO:The 1-2 residue removed from N-terminal for 3.There is no insertion in folding or lack.

Table 1：The 40 kinds of complete Leviviridae coat proteins retrieved from overall protein matter sequence library UniProt Sequence list

SEQ ID NO:341AQ3 enterobacteria phage MS2 coat protein T59S

ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYSIKVEVPKVAT QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:351GAV enterobacteria phage GA coat protein A59T G79V

ATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ VVNGVELPGSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIAEAISSQSGFYA

SEQ ID NO:361FRS enterobacteria phage FR coat proteins

>Sp | P03614 | shells _ BPFR coat protein OS=enterobacteria phage frPE=1 SV=4

ASNFEEFVLVDNGGTGDVKVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSANNRKYTVKVEVPKVAT QVQGGVELPVAAWRSYMNMELTIPVFATNDDCALIVKALQGTFKTGNPIATAIAANSGIY

SEQ ID NO:372VTU enterobacteria phage MS2 coat protein covalent dimers

Sp | P03612 | shells _ BPMS2 coat protein OS=enterobacteria phage MS2PE=1 SV=2

ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYANFTQFVLVDNGGTGDV TVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIF ATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:38(SEQ ID NO:38) G4WZU0G4WZU0_BPMS2116 enterobacterias phage ms2329852

>Sp | P03612 | shells _ BPMS2 coat protein OS=enterobacteria phage MS2 PE=1SV=2

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNLELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:39

D0U1D6 D0U1D6_BPMS2116 enterobacterias phage ms2 12022

>Tr | D0U1D6 | D0U1D6_BPMS2 coat protein OS=enterobacteria phage MS2 PE=4SV=1

SEQ ID NO:40

12022 gene ms2g2 of C0M2U4 C0M2U4_BPMS2116 enterobacterias phage ms2

>Tr | C0M2U4 | C0M2U4_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVELPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:41

12022 gene ms2g2 of C0M2S8 C0M2S8_BPMS2116 enterobacterias phage ms2

>Tr | C0M2S8 | C0M2S8_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNVELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:42

12022 gene ms2g2 of C0M212 C0M212_BPMS2116 enterobacterias phage ms2

Tr | C0M212 | C0M212_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV= 1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNPDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:43

12022 gene ms2g2 of C0M1M2 C0M1M2_BPMS2116 enterobacterias phage ms2

>Tr | C0M1M2 | C0M1M2_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTQFVLVDNGGTGDVAVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:44

12022 gene ms2g2 of C0M2L4 C0M2L4_BPMS2116 enterobacterias phage ms2

>Tr | C0M2L4 | C0M2L4_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVXQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:45

12022 gene ms2g2 of C0M220 C0M220_BPMS2116 enterobacterias phage ms2

>Tr | C0M220 | C0M220_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTXFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:46

Q2V0S8 Q2V0S8_BPBO1116 enterobacterias phage bo1 12014

>Tr | Q2V0S8 | Q2V0S8_BPBO1 coat protein OS=enterobacteria phage BO1 PE=4SV=1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGPLKDGNPIPSAIAANSGIY

SEQ ID NO:47

12022 gene ms2g2 of C0M216 C0M216_BPMS2116 enterobacterias phage ms2

>Tr | C0M216 | C0M216_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTQFVLVDNDGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:48

12022 gene ms2g2 of C0M1Y0 C0M1Y0_BPMS2116 enterobacterias phage ms2

>Tr | C0M1Y0 | C0M1Y0_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTQFVLVDNGGTGXVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:49

12022 gene ms2g2 of D0U1E4 D0U1E4_BPMS2116 enterobacterias phage ms2

>Tr | D0U1E4 | D0U1E4_BPMS2 coat protein OS=enterobacteria phage MS2 PE=4SV=1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNLELTIPIFATNPDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:50

12022 gene ms2g2 of C0M309 C0M309_BPMS2116 enterobacterias phage ms2

>Tr | C0M309 | C0M309_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPISSAIAANSGIY

SEQ ID NO:51

12022 gene ms2g2 of C0M325 C0M325_BPMS2116 enterobacterias phage ms2

>Tr | C0M325 | C0M325_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNVELTIPIFATNSDCEXIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:52

Q9T1C7 Q9T1C7_BPMS2116 enterobacterias phage ms12 110679

>Tr | Q9T1C7 | Q9T1C7_BPMS2 coat protein OS=enterobacteria phage M12 PE=4SV=1

MASNFTQFVLVDNGGTGDVTVXPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCALIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:53

12022 gene ms2g2 of C0M2Z1 C0M2Z1_BPMS2116 enterobacterias phage ms2

>Tr | C0M2Z1 | C0M2Z1_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=4SV =1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNVELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIX

SEQ ID NO:54

12022 gene ms2g2 of C0M1N8 C0M1N8_BPMS2116 enterobacterias phage ms2

SEQ ID NO:55

12022 gene ms2g2 of J9QBW2 J9QBW2_BPMS2116 enterobacterias phage ms2

>Tr | J9QBW2 | J9QBW2_BPMS2 capsid protein matter OS=enterobacteria phage MS2 PE=4SV=1

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVQLPVAAWRSYLNMELTIPIFATNDDCALIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:56

C8XPC9 C8XPC9_BPMS2113 enterobacterias phage ms2 329852

>Tr | C8XPC9 | C8XPC9_BPMS2 coat protein OS=enterobacteria phage MS2 PE=4SV=1

SEQ ID NO:57

12022 gene ms2g2 of C0M2Y4 C0M2Y4_BPMS2115 enterobacterias phage ms2

>Tr | C0M2Y4 | C0M2Y4_BPMS2 coat proteins (fragment) OS=enterobacteria phagies MS2GN=MS2g2PE =4SV=1

NFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQT VGGVELPVAAWRSYLNVELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:58

P69171 shells _ BPZR115 enterobacterias phage zr 332942

>Sp | P69171 | shells _ BPZR coat protein OS=enterobacteria phagies ZRPE=1SV=1

ASNFTQFVLVNDGGTGNVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:59

P69170 shells _ BPR17116 enterobacterias phage r17 12026

>Sp | P69170 | shells _ BPR17 coat protein OS=enterobacteria phage R17PE=1 SV=1

SEQ ID NO:60

P03612 shells _ BPMS2 enterobacterias phage ms2 329852

>Sp | P03612 | shells _ BPMS2 coat protein OS=enterobacteria phagies MS2PE=1SV=2

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:61

C0M1L4 C0M1L4_BPMS2116 enterobacteria phage ms212022 gene ms2g2

>Tr | C0M1L4 | C0M1L4_BPMS2 coat protein OS=enterobacteria phagies MS2GN=MS2g2PE=2SV =1

SEQ ID NO:62

329852 gene cp of C8XPD7 C8XPD7_BPMS2116 enterobacterias phage ms2

>Tr | C8XPD7 | C8XPD7_BPMS2 coat protein OS=enterobacteria phage MS2 GN=cpPE=4SV=1

SEQ ID NO:63

Q2V0T1 Q2V0T1_BPZR116 enterobacterias phage zr 332942

>Tr | Q2V0T1 | Q2V0T1_BPZR coat protein OS=enterobacteria phage ZR PE=4SV=1

SEQ ID NO:64

Q9MCD7 Q9MCD7_BPJP5115 enterobacterias phage jp501 12020

>Tr | Q9MCD7 | Q9MCD7_BPJP5 coat protein OS=enterobacteria phage JP501 PE=4SV=1

MASNFTEFVLVDNGETGNVTVAPSNFANGVAEWISSDSRSQAYKVTCSVRQSSAQNRKYTIKVAVPKVA TQTVGGVELPVAAWRSYLNMELTIPIFATNSDCALIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:65

P03611 shells _ BPF2115 enterobacterias phage f2 12016

>Sp | P03611 | shells _ BPF2 coat protein OS=enterobacteria phage f2 PE=1 SV=1

ASNFTQFVLVNDGGTGNVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT QTVGGVELPVAAWRSYLNLELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

SEQ ID NO:66

P34700 shells _ BPJP3115 enterobacterias phage jp34 12019

>Sp | P34700 | shells _ BPJP3 coat protein OS=enterobacteria phage JP34PE=3 SV=2

MATLRSFVLVDNGGTGDVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVT QVVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIADAISSQSGFYA

SEQ ID NO:67

Q2V0U0 Q2V0U0_BPBZ1115 enterobacteria phagies jp500332939

>Tr | Q2V0U0 | Q2V0U0_BPBZ1 coat protein OS=enterobacteria phage JP500 PE=4SV=1

SEQ ID NO:68

Q2V0T7 Q2V0T7_BPBZ1115 enterobacterias phage sd 332940

>Tr | Q2V0T7 | Q2V0T7_BPBZ1 coat protein OS=enterobacteria phage SD PE=4SV=1

MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVT QVVNGVELPISAWKAYASIDLTIPIFAATDDVTTISKSLAGLFKVGNPIADAISSQSGFYA

SEQ ID NO:69

Q9MBL2 Q9MBL2_BPKU1115 enterobacterias phage ku1 12021

>Tr | Q9MBL2 | Q9MBL2_BPKU1 coat protein OS=enterobacteria phage KU1 PE=4SV=1

MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVT QSVNGVELPVSAWKAFASIDLTIPIFAATDDVTLISKSLAGLFKIGNPVADAISSQSGFYA

SEQ ID NO:70

P07234 shells _ BPGA115 enterobacterias phage ga 12018

>Sp | P07234 | shells _ BPGA coat protein OS=enterobacteria phagies GAPE=1SV=3

MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYAIKLEVPKIVT QVVNGVELPGSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIAEAISSQSGFYA

SEQ ID NO:71

C8YJG7 C8YJG7_BPBZ1115 enterobacterias phage bz13 329853

>Tr | C8YJG7 | C8YJG7_BPBZ1 capsid protein matter OS=enterobacteria phage BZ13 PE=4SV=1

MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVT QVVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIAEAISSQSGFYA

SEQ ID NO:72

C8YJH1 C8YJH1_BPBZ1115 enterobacterias phage bz13 329853

>Tr | C8YJH1 | C8YJH1_BPBZ1 capsid protein matter OS=enterobacteria phage BZ13 PE=4SV=1

MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVT QTVNGVELPVSAWKAYASIDLTIPIFAATDDVTLISKSLAGLFKIGNPVADAISSQSGFYA

SEQ ID NO:73

C8YJH5 C8YJH5_BPBZ1115 enterobacterias phage bz13 329853

>Tr | C8YJH5 | C8YJH5_BPBZ1 capsid protein matter OS=enterobacteria phage BZ13 PE=4SV=1

MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVT QVVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGDPIADAISSQSGFYA

SEQ ID NO:74

Q2V0T4 Q2V0T4_BPTH1115 enterobacterias phage th1 12029

>Tr | Q2V0T4 | Q2V0T4_BPTH1 coat protein OS=enterobacteria phage TH1 PE=4SV=1

MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVT QVVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIADAISSQSGFYA

SEQ ID NO:75

Q2V0U3 Q2V0U3_BPBZ1116 enterobacterias phage tl2 332938

>Tr | Q2V0U3 | Q2V0U3_BPBZ1 coat protein OS=enterobacteria phage TL2 PE=4SV=1

SEQ ID NO:76

P03614 shells _ BPFR116 enterobacterias phage fr 12017

>Sp | P03614 | shells _ BPFR coat protein OS=enterobacteria phage frPE=1 SV=4

MASNFEEFVLVDNGGTGDVKVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSANNRKYTVKVEVPKVA TQVQGGVELPVAAWRSYMNMELTIPVFATNDDCALIVKALQGTFKTGNPIATAIAANSGIY

SEQ ID NO:77

Ef108465 enterobacterias phage r17 329852

Gi | 132424616 | gb | ABO33465.1 | coat proteins [enterobacteria phage MS2]

SEQ ID NO:781QBE

>Sp | P03615 | shells _ BPQBE coat protein OS=enterobacteria phage Qβ PE=1SV=2

AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQ NPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Additionally, amino acid residue is distinguished by the characteristic of its side chain.Their shared common backbones and the Conformation of the main chain for allowing Common set (Kleywegt ＆ Jones, Structure 4 1395-1400 (1996)), except two make an exception.Glycine The Conformation of the main chain not allowed to other aminoacid can be stably folded into, because its side chain is made up of single hydrogen atom.Proline Into stiffening ring, which is covalently bond to its main chain nitrogen by eliminating its acylamino hydrogen to side chain cyclisation, so that proline is just constrained in Conformation of the main chain small subset for other aminoacid, and eliminate its ability for becoming hydrogen bond donor.

Domain for being assembled into capsid is folded and domain combines (such as SEQ ID NO:3 aminoacid sequence leads to Cross following being stablized：Limit the backbone hydrogen bond syntype of its secondary structural unit, together side chain and stabilizing local structure or knot Close the hydrogen nearby between the backbone atoms of secondary structural unit (such as spiral, folding stock, curling, ring, corner and flexible end) Key, stabilizing local structure or combines neighbouring secondary structural unit and (such as spiral, folds stock, curling, ring, corner and flexibility together End) different side chains atom between hydrogen bond, and the close packing of hydrophobic side chain atom, the atom are acted on by model Moral China interact stably fold on energy and prevent solvent penetration to fold in, the solvent penetration may cause unstability with Local unfolding.The side chain of remaining residue is not involved in domain and folds maintenance or domain-domain interaction.If they Conformation of the main chain have only by Gly or cis Pro meet specialized requirements, so as to participate in domain folding, these residues are just Individually or as a group mutation, and the total topology on the folding of final structure domain or its surface can be had no substantial effect on, and can be led to Cross and following be clearly accredited as classification：Surface accessibility calculating is performed to known structure and (see, for example, Fraczkiewicz ＆ Braun,JMB；Meth Enzym；J Comp Chem 19,319 (1998)), subsequently the hydrogen bond for known structure is analyzed, albumen All routine techniquess in the research of matter 26S Proteasome Structure and Function.

It is used to check using two kinds of MS2 capsid structures from Protein Data Bank, is merged (with shape with N-terminal by C-terminal Into 2 domain protein white matter of single chain protein matter) the RNA containing stable octahedra capsid that formed of 2 MS2 capsid protein matter monomers With the 1AQ3 of the icosahedral capsid of 2VTU, 17 residues of identification (Ala1, Ser2, Thr5, Gln6, Ala21, Ala53, Val67, Thr69, Thr71, Val72, Val75, Ser99, Glu102, Lys113, Asp114, Gly115, Tyr129), its tool There are side chain positions (the Fraczkiewicz ＆ Braun server http of height solvation://curie.utmb.edu/ Getarea, using 1.4A solvent probes, without gradient, 2 region/energy/residues)；It is not involved in the hydrogen with the other parts of capsid Key (widely used freeware visual software bag Chimera (ERICF.PETTERSEN, THOMAS D.GODDARD, CONRAD C.HUANG,GREGORY S.COUCH,DANIELM.GREENBLATT,ELAINE C.MENG,THOMAS The hydrogen bond calculated in E.FERRIN (2004 J Comp Chem 25,1605-1612), with lax 0.5A and 30 degree hydrogen bond Standard)；And the Conformation of the main chain is allowed by all amino acid residues in addition to proline.When this 17 residues subset with When the structure alignment of enterobacteria phage MS2 compares, wherein ignoring GA and FR capsid sequences and in enterobacteria phage GA Or the residue being mutated in FR capsid sequences, stay and estimate 6 positions sensitive to mutation, and do not affect the structure or work(of monomer Can or its be assembled into the ability of stable capsid.This represents and wild type enterobacteria phage MS2 capsid protein matter (SEQ ID NO:3) 52% sequence iden.

In Secondary structural elements (spiral, folding stock, the corner with restriction hydrogen bond syntype and structuring ring such as Ω Ring) in residue insertion and/or disappearance promote these elements to lose which to limit hydrogen bonding or hydrophobic pack mode, or force its hydrogen Change in bonding or hydrophobic pack mode, this can change stability from urporotein sequence, shape and/or function. This can destroy packaging and affect the general stability for folding.On the other hand, expose with surface but the residue to protein folding Part do not provide it is crucial stable (generally via side chain for structural elements packaging, or proximity structure element is mutual Shielding of the acting surface with solvent or in the case of capsid with goods) destructuring ring, random coil and N and C-terminal be Following splendid candidates：(1) if the notable repositioning of the structural elements that need not connect, residue deletions, (2) if The addition of residue do not significantly change fold in structural elements relative disposal, or in the environment of protein by meet its with The exposed residue in hydrogen bonding capability screening surface of hydrogen bond donor or receptor, then the insertion of amino acid residue, or (3) naturally occurring One or more amino acid mutation or to non-natural residues, (which can be covalently attached to useful part, for example fluorogen, phosphorescent Group, Polyethylene Glycol, affinity tag, reporter group etc.) one or more mutation incorporation.Certainly, such insertion, disappearance and Mutation can occur in single appropriate members simultaneously or with any combinations, and its incorporation can cause with the albumen for improving feature Matter.The straightforward procedure for distinguishing insertion and/or the optimum for lacking is to be inserted into and/or lack scanning the multiple of sequence that be closely related Compare.In addition to N-terminal and C-terminal addition and lacking, it is known that Leviviridae coat protein sequence does not have for each other Insertion or disappearance.This is not intended to not insert and/or lack.The knot of the remoter member of folding family is had to check for simply Structure/function.

Simplest multiple alignment algorithm generally can be obtained to general public at public domain sequence and structural database.Such as Infructescence column space by from high % homogeneity such as 90% to the continuous sequence of low % homogeneity such as 20% spectrum fill, then these Algorithm can correctly compare the sequence of shared extremely low % homogeneity.When these clusters share low % homogeneity, these algorithms tend to not The sequence cluster with identical folding can correctly be compared；If however, the x-ray crystal structure of one or more members of each cluster It is resolved and fully refines, then such cluster successfully and clearly can be compared.Tied by two grades of best overlay protein structure The backbone atoms of constitutive element part, the protein are closely related but by the remote correlation of sequence, are clearly limited to its sequence by folding One-to-one correspondence between row, and the high % homogeneity cluster being successfully generated by simple sequence alignment schemes can be anchored into Pairing due to main chain superposition is compared, and the correct overall sequence of the folding family for generating is compared, so as to cause to fold family Member the significant comparison of topology (Arthur M Lesk, Michael Levitt, Cyrus Chothia (1986), ProtEng1,77-78).By checking that overall sequence is compared, can check Comprehensive picture of its form of evil or function.

Alloleviviridae coat proteins belong to and Leviviridae coat protein identical folding family (folding Folded family is d.85.1), and be also fitted with into by the icosahedral capsid of 180 monomer compositions.Show in deck watch in Figure 27 The multiple alignment of the alloleviviridae coat protein sequences of preservation in UniProt.60 (60%) percent Alloleviviridae coat protein sequences are conservative.The coat protein of Leviviridae and alloleviviridae 130 aminoacid are about, but because identical residue percentage ratio is very low, about 20%, so Multiple sequence alignments algorithm is generally not Leviviridae sequence alignment alloleviviridae can be correctly directed to.Realize that this straightforward procedure put is to reverse sequence, and And subsequently reversed sequence is compared using same approach.The multiple alignment of sequence and reversed sequence will be inconsistent.This difficulty can be led to Cross inspection representative structure to be avoided.allolevividae Qβ(PDB-ID:1QBE)(SEQ ID NO:78, see below) clothing The x-ray crystal structure of shell has been preserved in public regional data base RCSB Protein Data Bank (http://www.rcsb.org) in. Compare instrument using the jFATCAT at the RCSB Protein Data Banks, dropped to by making the root-mean-square-deviation between { C α } atom It is minimum, make independent refined 1QBE monomers be fitted to independent refined 1AQ3 monomers.Root-mean-square-deviation is in scope 2.33-2.76 angstrom In, this depends on which independently refined monomer compared, mainly due to connect the N-terminal residue 1-3 of Secondary structural elements with And the main chain of section 8-18,26-28,50-55 and 67-76 (numbering is with reference to the residue of equal value of the topology in MS2 structures 1AQ3) Difference in disposal, as shown in Figure 22-25 and described in description of the drawings.Due to the conformational difference in same area, for The monomer that independence in 1AQ3 is refined, is 1.72A by the main chain root-mean-square-deviation that jFATCAT is measured.Topology is compared and is shown In table, for 1AQ3 indicates to specify (DSSP, W Wolfgang Kabsch by the secondary structure of hydrogen bond syntype Christian Sander (1983), Biopolymers22,2577-2636), and or because refined Conformation of the main chain is base It is different in sheet, or be not limited in electron density in subtractive process and show very big deviation because section is easily moved very much Section is provided with lower case.The region for showing backbone flexibility in crystalline environment is also the splendid candidate for inserting and/or lacking Thing, as the interaction between these residues and the remainder of folding is stably important, its electron density for folding By localization.Identical information is added to 2VTU and is provided and be most suitable for adapting to further appreciating that for the section for changing.These compare in figure Symbolic capture in 26, described Figure 26 show 1AQ3 relative to 2VTU relative to 1QBE comparison.

The inspection of 1AQ3 and 1QBE monomers provides following understandings, such as further by reference to Figure 28-31 and its each self-described Illustrate.All residue numberings are given for the monomer in 1AQ3.

This still means that the sequence relative to 1QBE enterobacteria bacteriophage coat protein matter Q β, SEQ ID#3 enterobacteria phagies The folding of MS2 coat proteins is preserved down to 21% homogeneity, and for referred herein to all alloleviviridae outside Glutelin matter sequence, is 16% homogeneity for conserved residues.In the side chain positions of the relatively early height solvation for calculating only One, side chain is not involved in the hydrogen bond with the other parts of capsid and its Conformation of the main chain is by all aminoacid in addition to proline Residue is allowed, and keeps conservative, Y129 (in SEQ NO 3 are numbered).Its backbone locations and side chain are packaged in by the MS2 for merging Substantially change in the octahedra enterobacteria phage MS2 capsid structure that dimer (2VTU) is formed.Consider that the last change makes Threshold amino acid Percentage of sequence identity reaches 15%.Referring to the deck watch in Figure 26 and Figure 27, (1AQ3 is relative to 2VTU phases For 1QBE, and for the allolevi Multiple sequence alignments tables of explanation).All Similarity Percents in the paragraph are only It is effective in the case where Structure anchor is compared.

N-terminal residue 1-3 can meet its hydrogen bonding potentiality with C-terminal residue 129 and water, and vice versa；Therefore, Some or all in these residues should be able to be lacked, and stable VLP is formed by the protein of truncate.Figure 32 is displayed in assembling Capsid in the non-covalent dimeric main chain colour band diagram of 3 of point packaging symmetrical about non-covalent enterobacteria phagies MS2.Institute There is N-terminal color to be green, C-terminal color is redness.The close sequence for meaning monomer of end can be fused into it is single-stranded, with Covalent dimer is formed, as by a monomer is added after another for 2VTU is completed, that is, produced by (monomer residue 1- 129- monomer residue 1-129) the wall scroll protein chain that constitutes, or by adding other connection residue between sequence monomer (monomer 1-129-linker residue-monomer 1-129), as long as relative chain direction (from N-terminal to C-terminal) is allowed by the Series Sheet bodily form Into continuous peptide chain.The monomer of linker residue-monomer series-connected need not be added and be resolved (PDB-ID:2VTU).In 2VTU, each Non-covalent dimer has been transformed into single protein；However, because the C α of residue 2 and 129 are separated by about 6 angstroms, reluctantly enough Closely to be connected with jointing, and folding is not upset (C α-C α distance restraints in about 3.8 angstroms, due to the resonance shape of peptide unit Formula), and in some monomers, their main chain hydrogen bonding each other.The β lamellas side shape of each dimer (covalently or non-covalently) Into the inwall of capsid.The geometry of β lamellas can be limited by the curvature of lamella (Cyrus Chothia, Jiri Novotny, Robert Bruccoleri,Martin Karplus(1985)J Mol Biol 186,651-663).Tight idol in 2VTU Connection makes β lamellas be constrained to relatively low curvature, so as to cause octahedron rather than icosahedral capsid.0-6 is mixed between monomer individual residual The joint of base by provide allow covalent dimer relax into icosahedral capsid it is identical needed for sufficiently flexible, wherein physical property May more be closely related with the non-covalent capsid structure of icosahedron.Usually, joint will be 1-6 residue.However, for example, The covalent dimer of 2VTU is of virtually the Ser2 lacked in the second copy.In such cases, joint length can be 0。

Select the residue for joint there be little side chain, caused with avoiding relatively smaller volume being packaged into by a large amount of atoms Steric strain.Tension force can be also dropped to by avoiding amino acid residue such as Pro of the selection with less Conformation of the main chain space It is minimum.Avoid tension force from being transformed into the protein for more rapidly or more effectively folding.Particularly in the centre portion of longer ring Huger and charged side chain tends to becoming the combination target of protease.Joint containing Gly is preferred.

According to Figure 32 also it is clear that the C-terminal of a monomer may be connected to non-covalent dimeric monomer near participation N-terminal, and stable icosahedral capsid can be still formed, if joint has suitable length and flexibility, and in capsid ring Without the potential cleavage site that can be close to by protease in border.In fact, three monomers can be connected and still be formed by appropriate joint The shell section.Because the yellowish-brown of Figure 32, Lycoperdon polymorphum Vitt and middle blue monomer are also the asymmetry unit of capsid.With appropriate connection Three monomers of the end of section to terminal tandem should be able to also form stable icosahedral capsid.

N-terminal residue 1-3 can meet its hydrogen bonding potentiality with C-terminal residue 129 and water, and vice versa；Therefore, Should be able to lack some or all in these residues, and the protein by truncate or alternately by by series protein matter In the corresponding potential joint length that extends of disappearance number, form stable VLP.

Correspondingly, present disclosure includes the VLP comprising capsid, and the capsid includes such capsid protein matter, and which is Wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) variant, and to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 Hydrolysis be resistance.For example, VLP can include such capsid protein matter, and which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, in addition to being lacked in the A residues at the 1st.VLP can include such capsid Protein, which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, except at the 1st A residues lacked and outside the S residues at the 2nd are lacked.VLP can include such capsid protein matter, and which has open country Raw type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, lacked except the A residues at the 1st, Outside the S residues at the 2nd are lacked and N residues at the 3rd are lacked.VLP can include such capsid protein matter, Which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, except the Y at the 129th it is residual Outside base is lacked.VLP can include such capsid protein matter, and which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but with single (1) aminoacid deletion in 112-117 sections.VLP can include such Capsid protein matter, which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but with Single (1) aminoacid deletion in 112-117 sections.VLP can include such capsid protein matter, and which has wild type intestinal bar Bacterium phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but with the 1-2 residue insertion in 65-83 sections, And the hydrolysis to being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is resistance.VLP can include such capsid protein matter, its tool There are wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but with the 1-2 in 44-55 sections Individual residue insertion.VLP can include such capsid protein matter, and which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but with single (1) the residue insertion in 33-43 sections, and to by peptide bond hydrolysis enzyme The hydrolysis of the other catalysis of EC 3.4 is resistance.VLP can include such capsid protein matter, and which has wild type enterobacteria phage MS2 capsids (SEQ ID NO:3) aminoacid sequence, but with the 1-2 residue insertion in 24-30 sections.VLP can be included Such capsid protein matter, which has wild type enterobacteria phage MS2 capsid (SEQ ID NO:3) aminoacid sequence, but With single (1) the residue insertion in 10-18 sections.VLP can be comprising the capsid egg connected with the second capsid sequence monomer White matter sequence monomer, the capsid protein matter sequence monomer is assembled into the hydrolysis being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 is The capsid of resistance.VLP can include the capsid protein matter sequence monomer that its C-terminal is extended by 0-6 residue linkers section, the joint The C-terminal of section is connected with the second capsid sequence monomer, all these to be assembled into being catalyzed by the other EC of peptide bond hydrolysis enzyme 3.4 Hydrolysis be resistance capsid.Suitable joint sequence including but not limited to-(Gly) x-, wherein x is 0-6, or Gly-Ser connects Head is such as, but not limited to-Gly-Gly-Ser-Gly-Gly- ,-Gly-Gly-Ser and-Gly-Ser-Gly-.VLP can also comprising with The capsid protein matter sequence monomer of the 3rd capsid sequence monomer series connection, the capsid protein matter sequence monomer are assembled into by peptide bond The hydrolysis of the catalysis of hydrolytic enzyme classification EC 3.4 is the capsid of resistance.Again, in capsid protein matter, C-terminal can be connect by 0-6 residues Head sections extend, and the C-terminal of the joint section is connected with the 3rd capsid sequence monomer, all these to be assembled into by peptide bond water The hydrolysis of the other catalysis of EC 3.4 of solution enzyme is the capsid of resistance.One or two joint sequences may be selected from-(Gly) x-, wherein x= 0-6, or selected from the Gly-Ser joints of-Gly-Gly-Ser-Gly-Gly- ,-Gly-Gly-Ser and-Gly-Ser-Gly-.Example Such as, in one or two joint sequences, joint is-(Gly) x-, and x is 1,2 or 3.VLP can include its N-terminal truncate 1- One or more coat protein sequence of 3 residues, wherein joint sequence extend the number of residues of disappearance, wherein joint sequence It is-(Gly) x-, wherein x=0-6.For example, VLP can include one or more coat protein of its 1 residue of C-terminal truncate Sequence, and subsequently joint sequence extends 1 residue, and wherein joint sequence is-(Gly) x-, wherein x=0-6.VLP can be included Two kinds of coat protein sequences, wherein first 1 residue of coat protein sequence C end truncate in series connection dimer, and And joint sequence extends 1 residue, or first and/or second housing protein sequence C-terminal wherein in series connection trimer 1 residue of truncate, wherein joint sequence are-(Gly) x-, wherein x=0-6.

The disclosures of all patents and publication include that those being listed herein below are incorporated hereby this Text.

List of references

1.Shenton, W. et al. (2001) Synthesis of Nanophase Iron Oxide in Lumazine Synthase Capsids.AngewandteChemie.113:456-459

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Claims

1. it is a kind of for purification enclose at least one heterologous cargo molecule, by SEQ ID NO:Enterobacteria phage shown in 3 The method of the VLP of MS2 capsid proteins matter composition, methods described include：A () obtains the cell lysate comprising multiple VLP； B () makes the cell lysate that the time and condition that be enough to hydrolyze the product of cell lysis in addition to the VLP is contacted with protease, To form hydrolyzate；(c) VLP is separated from the hydrolyzate.

2. method according to claim 1, wherein the protease is the other EC of peptide bond hydrolysis enzyme 3.4.

3. method according to claim 1, wherein the protease is selected from E.C. 3.4.21.64, from the albumen of streptomyces griseuses Enzyme, the protease from Bacillus licheniformis, pepsin and papain.

4. method according to claim 1, wherein step (b) are performed about 30 minutes.

5. method according to claim 1, wherein step (b) are performed at about 37 DEG C.

6. method according to claim 1, wherein step (b) also include making the cell lysate and nuclease, starch At least one contact in enzyme and lipase.

7. method according to claim 1, wherein step (c) including making the hydrolyzate centrifugation, to obtain comprising pressing The precipitate of the VLP of weight meter at least 90%.

8. method according to claim 1, wherein step (c) perform the hydrolyzate using ammonium sulfate including (i) The first precipitation, be subsequently centrifuged for first, to obtain the first precipitate and the first supernatant；(ii) to first supernatant The second precipitation using ammonium sulfate is performed, is subsequently the second centrifugation, to obtain the second precipitate, wherein the second precipitate bag Containing by weight at least 90% VLP.

9. method according to claim 1, wherein step (c) perform the hydrolyzate using ethanol including (i) First precipitation, is subsequently the first centrifugation, to obtain the first precipitate and the first supernatant；(ii) first supernatant is held Second precipitation of enforcement ammonium sulfate, is subsequently the second centrifugation, to obtain the second precipitate, wherein second precipitate is included By weight at least 90% VLP.

10. method according to claim 1, wherein the heterologous cargo molecule comprising be coupled the heterologous cargo molecule and The oligonucleotide joint of the VLP.

11. methods according to claim 10, wherein the oligonucleotide joint is oligoribonucleotide.

12. methods according to claim 1, wherein the heterologous cargo molecule include selected from siRNA, shRNA, The oligoribonucleotide of sshRNA, lshRNA and miRNA.

13. methods according to claim 1, wherein the heterologous cargo molecule includes the few ribonucleotide comprising ribozyme Acid.

14. method according to claim 1, wherein the heterologous cargo molecule includes peptide or polypeptide.

15. methods according to claim 1, wherein the detached VLP is comprising for described from the hydrolyzate One or more product of cell lysis present in cell lysate per 100 grams of capsid protein matter less than 4 grams, wherein described thin Cellular lysate product is selected from protein, polypeptide, peptide and its any combinations.

16. methods according to claim 15, wherein it is described for hydrolysis time and condition be enough to cut the cell Present in lysate but not by the capsid enclose per 100 polypeptides at least 60, while the institute before such hydrolysis At least 60 in stating present in cell lysate per 100 capsids keep complete after the hydrolysis.

17. methods according to claim 15, wherein it is described for hydrolysis time and condition be enough to cut the cell Present in lysate but not by the capsid enclose per 100 polypeptides at least 70, while the institute before such hydrolysis At least 70 in stating present in cell lysate per 100 capsids keep complete after the hydrolysis.

18. methods according to claim 15, wherein it is described for hydrolysis time and condition be enough to cut the cell Present in lysate but not by the capsid enclose per 100 polypeptides at least 80, while the institute before such hydrolysis At least 80 in stating present in cell lysate per 100 capsids keep complete after the hydrolysis.

19. methods according to claim 15, wherein it is described for hydrolysis time and condition be enough to cut the cell Present in lysate but not by the capsid enclose per 100 polypeptides at least 90, while the institute before such hydrolysis At least 90 in stating present in cell lysate per 100 capsids keep complete after the hydrolysis.

20. method according to claim 1, wherein the heterologous cargo molecule is included comprising short rna, ribozyme and packaging sequence The oligoribonucleotide of row.

21. methods according to claim 1, wherein the heterologous cargo molecule include it is double comprising multi-functional polynucleotide Molecule cargo molecule, at least first fit sequence of the multi-functional polynucleotide comprising specifically binding bioactive molecule, With the second fit sequence for the packaging sequence with reference to the capsid.

22. methods according to claim 21, wherein the bioactive molecule includes herbicide or insecticide.

23. methods according to claim 1, wherein the cell lysate obtains self-contained first vector and Second support Cell, nucleotide sequence of the first vector comprising encoding proteins enzyme resistance capsid protein matter, the Second support include coding The nucleotide sequence of heterologous cargo molecule.

24. methods according to claim 1, wherein the cell lysate obtains the cell of self-contained carrier, the carrier The nucleotide sequence of the nucleotide sequence comprising encoding proteins enzyme resistance capsid protein matter and encoding heterologous cargo molecule.

25. methods according to claim 1, wherein the cell lysate derives from prokaryotic host cell.

26. methods according to claim 1, wherein the cell lysate derives from eukaryotic host cell.

A kind of 27. methods for protecting target molecule not hydrolyzed, methods described include：A () selects to be formed to by peptide bond hydrolysis The capsid protein matter of the capsid of the hydrolytic resistance of the other catalysis of EC 3.4 of enzyme, the capsid protein matter such as SEQ ID NO:Shown in 3； B () is with the carrier of the nucleotide sequence comprising the coding capsid protein matter and comprising the nucleotide sequence for encoding the target molecule Carrier, stable transfection host cell；C () maintains the host cell to be enough to make the inverted cell expression and assemble The time of the capsid of target molecule described in encapsidate is with the conditions of；Target described in the method purification encapsidate of (d) by claim 1 point The VLPs of son.